InteractiveFly: GeneBrief

Secretory 16: Biological Overview | References


Gene name - Secretory 16

Synonyms -

Cytological map position - 11B2-11B3

Function - scaffolding protein

Keywords - a component of the endoplasm stress granule formed in response to amino acid starvation, follicle cell differentiation, Clueless together with dGRASP prevents ER stress and therefore maintains Sec16 stability essential for the functional organization of perinuclear early secretory pathway

Symbol - Sec16

FlyBase ID: FBgn0052654

Genetic map position - chrX:12,595,984-12,609,906

NCBI classification - ACE1-Sec16-like: Ancestral coatomer element 1 (ACE1) of COPII coat complex assembly protein Sec16

Cellular location - cytoplasmic



NCBI links: EntrezGene
BIOLOGICAL OVERVIEW

Most cellular stresses induce protein translation inhibition and stress granule formation. Stress granules are well-studied, cytoplasmic reversible, pro-survival stress assemblies where untranslated free RNAs (resulting from protein translation inhibition) are stored and protected together with RNA-binding proteins, translation initiation factors, and the 40S ribosomal subunits. This study used Drosophila S2 cells to investigate the role of G3BP/Rasputin in this process. In contrast to arsenite treatment, where dephosphorylated Ser142 Rasputin is recruited to stress granules, this study found that, upon amino acid starvation, only the phosphorylated Ser142 form is recruited. Furthermore, Sec16, a component of the endoplasmic reticulum exit site (Tang, 2017; Sprangers, 2016), was identified as a Rasputin interactor and stabilizer. Sec16 depletion results in Rasputin degradation and inhibition of stress granule formation. However, in the absence of Sec16, pharmacological stabilization of Rasputin is not enough to rescue the assembly of stress granules. This is because Sec16 specifically interacts with phosphorylated Ser142 Rasputin, the form required for stress granule formation upon amino acid starvation. Taken together, these results demonstrate that stress granule formation is fine-tuned by specific signaling cues that are unique to each stress. These results also expand the role of Sec16 as a stress response protein (Aguilera-Gomez, 2017).

Stress granules are well-studied, cytoplasmic reversible, pro-survival stress assemblies where untranslated free RNAs (resulting from protein translation inhibition) are stored and protected together with RNA-binding proteins, translation initiation factors, and the 40S ribosomal subunits. Stress granule formation has been best investigated in mammalian cells upon different type of stresses, including heat and oxidative stress. This has led to the identification of a number of factors that are essential for their formation, such as the case for Tia-1 and Ras-GAP SH3 domain-binding protein (G3BP1/2, referred to as G3BP hereafter) (Aguilera-Gomez, 2017).

G3BP was first identified in human cells through co-immunoprecipitation with the SH3 domain of RasGAP. However, it has an RNA recognition motif (RRM) toward the C terminus, suggesting that it binds mRNAs. In growing cells, G3BP is normally cytoplasmic; However, after stress induction (especially stress leading to eIF2α phosphorylation), it is not only readily recruited to stress granules but also is necessary for their formation. Furthermore, G3BP drives stress granule formation when overexpressed in the absence of stress. Importantly, G3BP is phosphorylated on Ser149 during basal conditions, but it needs to be dephosphorylated to drive stress granule assembly triggered by arsenite treatment. Taken together, G3BP is critical for stress granule formation when its Ser149 is dephosphorylated and through its binding to Caprin and the 40S ribosomal subunit. Conversely, G3BP is inhibited when binding to peptidase USP10 (Aguilera-Gomez, 2017).

G3BP also appears to have an important role in disease. First, viruses can exploit G3BP. However, G3BP also appears to slow down HIV replication by leading to the sequestration of viral mRNA. Second, G3BP is overexpressed in gastric cancer and bone and lung sarcomas, where it is considered as a marker for poor survival. Strikingly, downregulation of G3BP in cells and in vivo reduces stress granule formation as expected but also tumor invasion and metastasis, showing a clear role for stress granule formation in cancer. Last, G3BP is a target of TDP-43 that is often mutated, mislocalized, and misaccumulated in amyotrophic lateral sclerosis (ALS) (Aguilera-Gomez, 2017).

Stress granules are also formed in Drosophila, for instance, upon heat stress, arsenite exposure (Farny, 2009), and amino acid starvation of Drosophila S2 cells (Zacharogianni, 2014). However, the mechanism behind their formation upon this latter stress is not completely understood. Interestingly, amino acid starvation leads to the formation of another recently described stress assembly, the Sec bodies that store and protect most of the COPII subunits and the endoplasmic reticulum exit site (ERES) component Sec16 (Zacharogianni, 2014). Importantly, Sec bodies and stress granules are independent structures that are formed at the same time frame of amino acid starvation (Aguilera-Gomez, 2017).

Sec16 is a conserved peripheral membrane protein that tightly localizes and concentrates to the ERES via a domain that has been mapped to a small arginine-rich region upstream of the conserved central domain. It binds nearly all COPII subunits and controls at least two aspects of COPII-coated vesicle dynamics (Sprangers, 2015). Sec16 is essential for endoplasmic reticulum (ER) to Golgi transport, especially in Drosophila where the absence of Sec16 results in a severe inhibition of protein exit from the ER (Aguilera-Gomez, 2017).

Interestingly, Sec16 responds to nutrient stress (Zacharogianni, 2011, Zacharogianni, 2014). In this regard, it has been shown that Sec16 is a key factor driving Sec body formation upon amino acid starvation that activates the ER-localized dPARP16. In turn, dPARP16 mono-ADP-ribosylates Sec16 on a conserved sequence close to its C terminus (Aguilera-Gomez, 2016), and Sec16 modification by dPARP16 is enough to elicit Sec body formation. This demonstrates that Sec16 is a stress response protein that plays an important role in the response to amino acid starvation (Aguilera-Gomez, 2017).

This study shows that the phosphorylation state of the G3BP Drosophila ortholog Rasputin (Rin) is differentially required for the formation of stress granules that are formed upon arsenite treatment and amino acid starvation. Whereas stress granule formation upon arsenite treatment requires the non-phosphorylated form of Rin (as is the case for G3BP in mammalian cells), amino acid starvation requires the phosphorylated form. Furthermore, Sec16 was shown to specifically interact with phosphorylated Rin and mediates this differential requirement (Aguilera-Gomez, 2017).

All together, these results provide a link between the protein transport from the ER and protein translation. It also explains the specific requirement of Sec16 for stress granule formation upon amino acid starvation, but not other stresses. Furthermore, it enlarges the scope of Sec16 function at the ER by identifying yet a new role in the response to amino acid starvation (Aguilera-Gomez, 2017).

Stress granules are formed when protein translation initiation is inhibited by cellular stress that leads to elF2α phosphorylation and the accumulation of untranslated mRNAs. Stress granule components start to coalesce through protein-protein interactions mediated by proteins containing regions of low-complexity sequences and displaying multivalence interactions. This process is facilitated by the presence of accumulating free mRNAs (Molliex, 2015, Patel, 2015). Accordingly, incubation of stressed cells with cycloheximide, which locks ribosomes to the mRNA, blocks stress granule formation, whereas puromycin stimulates their formation. Stress granule formation is also driven by a number of critical factors, including G3BP. In all these respects, the cytoplasmic foci that are formed in Drosophila cells upon amino acid starvation and that are positive for RNA-binding proteins are bona fide stress granules, as they share many features of those found in mammalian cells after arsenite treatment, heat stress, and ER stress (Aulas, 2017). Furthermore, this study shows that Rin, the G3BP Drosophila ortholog, is an essential factor for amino acid starvation-driven stress granules (Aguilera-Gomez, 2017).

However, stress granule formation displays a certain level of heterogeneity. First, some of the signaling cues inducing their formation appear to be different. For instance, although elF2α phosphorylation is required for arsenite-triggered stress granule formation in Drosophila cells, this phosphorylation is not necessary for their formation upon heat stress. Second, mammalian stress granules that are formed upon different stresses appear to have slightly different content, at least in HAP1 cells (Aulas, 2017). Third, stress granules display different material properties. Yeast stress granules possess a solid core made of components that exchange slowly and less dynamically, whereas mammalian stress granules have liquid droplet properties. This is also reflected by the fact that stress granules contain diverse proteomes (Aguilera-Gomez, 2017).

Remarkably, the phosphorylation status of Rin was shown to dictate its differential recruitment to stress granules formed upon arsenite treatment and amino acid starvation. Phosphorylated Rin (on Ser142) is instrumental for stress granule formation upon amino acid starvation, whereas arsenite-driven stress granules require dephosphorylated Rin (as for G3BP in mammalian cells). This is the first example that clearly demonstrates this differential usage. This further documents that stress granules are more complex and variable than previously anticipated and not just a temporal storage of stalled ribonucleoprotein particles (RNPs) (Aguilera-Gomez, 2017).

What determines the use for phosphoRin versus non-phosphoRin in stress granule formation upon different stresses is not fully understood, but the first clue is the discovery that the large hydrophilic ERES protein Sec16 specifically interacts with phosphoRin (Aguilera-Gomez, 2017).

Sec16 adds to the lengthening list of factors modulating stress granule formation via their interaction with G3BP, such as Caprin, TDP43, and Usp10. In addition, YB-1 promotes the translation of G3BP1 mRNA. Sec16 depletion leads to a reduced level of total Rin, but the remaining Rin pool is enough to lead to stress granule formation upon arsenite treatment, heat stress, and ER stress (Aguilera-Gomez, 2017).

Interestingly, Rin level is further reduced when Sec16-depleted cells are amino acid starved. Thus, Sec16 depletion upon amino acid starvation mimics Rin depletion, explaining the inhibition of stress granule formation upon this stress. This suggests that Sec16 protects Rin against proteasome degradation. It is very often the case that proteins in a complex stabilize each other and that when one partner is absent the other is degraded. However, Rin depletion does not affect Sec16 stability. Sec16 has, therefore, an active role in protecting Rin, perhaps by preventing Rin ubiquitination, a signal for degradation through the proteasome (Aguilera-Gomez, 2017).

However, although proteasome inhibition restores Rin level in starved Sec16-depleted cells, this is not enough to recover stress granule formation. This suggests that Sec16 has an additional role. Given that Sec16 specifically interacts with phosphoRin, this interaction appears necessary not only to protect and stabilize phosphoRin but also to facilitate the role of phosphoRin in stress granule formation upon amino acid starvation. It is possible that the Sec16/phosphoRin complex is recruited to stress granules and/or that Sec16 allows Rin to bind another stress granule partner. In any case, this is strictly specific for amino acid starvation (Aguilera-Gomez, 2017).

Indeed, phosphoRin is the form that is specifically required for stress granule formation upon amino acid starvation. Upon arsenite treatment, the dephosphorylated form of Rin is the form required for stress granule formation both in Drosophila (current results and in mammalian cells; Kedersha, 2016, Tourriere, 2003). This is mirrored by the role of Sec16 that is specifically required for stress granule formation upon amino acid starvation, but not upon heat stress, ER stress, and arsenite treatment. Accordingly, Sec16 does not interact with dephosphorylated Rin (Aguilera-Gomez, 2017).

Does Sec16 have a role in Rin phosphorylation? Rin phosphorylation could take place in the cytoplasm independently of Sec16 that would then recognize phosphoRin and stabilize it. Alternatively, Sec16 could contribute to Rin phosphorylation by acting as a scaffold for the kinase required for Rin phosphorylation. This kinase, which is likely to be (hyper-)activated by amino acid starvation, remains to be identified (Aguilera-Gomez, 2017).

Why does amino acid starvation require this specific Sec16/phosphoRin interaction? Why is an ERES component functionally linked to stress granule formation specifically upon this stress? It is likely that this specific interaction elicits the formation of unique stress granules, perhaps storing mRNAs encoding proteins key for survival upon starvation and fitness upon stress relief. In this respect, stress granules formed during amino acid starvation contain the P-body component Tral that is not found in those formed upon heat shock (Jevtov, 2015). Interestingly, Tral has been shown to bind mRNAs encoding COPII subunits, and it is possible that other mRNAs encoding or secretory pathway components may be sequestered and protected inside these stress granules. This would reflect a type of multiplexing that has been observed in neurons. Conversely, as ER-translated mRNAs (possibly encoding secretory proteins required in stress recovery) are proposed to escape sequestration to stress granules , Sec16 interaction with stress granule components might restrict stress granule formation to specific sites away from these mRNAs (Aguilera-Gomez, 2017).

Last, the enrichment of phosphoRin (and the presence of Sec16) in amino acid starvation-driven stress granules might change their material properties and, consequently, their dynamics. This is suggested by the large size of the S142E-positive stress granules and by their poor reversibility. Consequently, the exchange of components with the surrounding cytoplasm might be reduced in phosphoRin-based stress granules when compared to stress granules that depend on the dephosphorylated form of Rin (and G3BP). This is supported by fluorescence recovery after photobleaching (FRAP) experiments. G3BP-positive arsenite-driven stress granules show full recovery in less than 1 s, whereas the recovery of stress granules formed upon amino acid starvation is an order of magnitude slower. The biological relevance of this difference is, however, not fully understood (Aguilera-Gomez, 2017).

Overall, the results presented in this study are in congruence with evidence of the link among protein translation, RNA metabolism, and the secretory pathway. Stress granules are formed in response to ER stress. P-bodies also localize in close proximity to the ER and increase in number in response to ER homeostasis perturbations and in Arf1 yeast mutant. Last, ER-resident proteins are shown to regulate P-body formation in yeast (Aguilera-Gomez, 2017).

The results provide further evidences of the versatility of Sec16. In growing conditions, mammalian Sec16 exists as two isoforms that are both localized to the ERES but have non-redundant functions in humans. Whereas Sec16A is classically required for the ER exit of proteins destined to the Golgi and the plasma membrane, Sec16B specializes in transport to peroxisomes. Furthermore, Sec16 exons have been shown to be alternatively spliced upon T cell activation, and increased expression of the Sec16 isoform containing exon 29 leads to an increased number of ERESs and more efficient COPII transport in activated T cells. In this regard, Sec16 is also specifically phosphorylated by ERK2 upon serum stimulation in mammalian cells, leading to an increase in the number of ERESs and a larger secretory capacity. Sec16 also interacts with LKKR2, albeit in a kinase activity-independent fashion, and with ULK (Atg1) in non-stressed conditions (Aguilera-Gomez, 2017).

Sec16 also plays key roles in the response to stress, for instance, to ER stress where it appears to mediate the Golgi bypass of transmembrane proteins (Piao, 2017), but also to nutrient stress (Zacharogianni, 2011). Amino acid starvation is an interesting stress as it triggers the formation of two stress assemblies in the same time frame, both requiring Sec16 but in two different manners: The first, the MARylation of Sec16 on its C terminus by ER-localized dPARP16, is an event that is enough to trigger the formation of Sec bodies (Aguilera-Gomez, 2016). The second is the Sec16 interaction and stabilization of phosphoRin, leading to the formation of stress granules. Interestingly, neither of these is linked to the Sec16 role in protein exit from the ER or COPII-coated vesicle dynamics (Zacharogianni, 2014; Aguilera-Gomez, 2017 and references therein).

Taken together, this demonstrates the versatility and capacity of the large scaffold protein Sec16 to regulate very diverse cellular processes, many of them pro-survival. Therefore, more Sec16 interactors need to be identified and studied (Aguilera-Gomez, 2017).

In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation

PARP catalysed ADP-ribosylation is a post-translational modification involved in several physiological and pathological processes, including cellular stress. In order to visualise both Poly-, and Mono-, ADP-ribosylation in vivo, specific fluorescent probes were engineered for this study. Using them, amino-acid starvation was shown to trigger an unprecedented display of mono-ADP-ribosylation that governs the formation of Sec body, a recently identified stress assembly that forms in Drosophila cells. dPARP16 catalytic activity is necessary and sufficient for both amino-acid starvation induced mono-ADP-ribosylation and subsequent Sec body formation and cell survival. Importantly, dPARP16 catalyses the modification of Sec16, a key Sec body component; this modification was shown to be a critical event for the formation of this stress assembly. Taken together these findings establish a novel example for the role of mono-ADP-ribosylation in the formation of stress assemblies and link this modification to a metabolic stress (Aguilera-Gomez, 2016).

By using biological modules known as macrodomains that do not posses any hydrolase activity, a specific and stable Mono-ADP-ribosylation detection probe (MAD) that has important specifications was built and optimised: First, MAD specifically detects and binds Mono-ADP-ribose. This specificity is sustained by three arguments: (1) MAD is designed and built using PARP14 macrodomains. Crystallography and calorimetric assays show that each macrodomain has a conserved fold with high binding affinity for mono-ADP-ribose (particularly macrodomains 2 and 3) and the affinity required for in vivo visualisation is provided by the three macrodomains together in a cooperative manner. (2) the mutated G1055E probe (affecting the binding pocket of macrodomain 2 in such as way that it does not bind mono-ADP-ribose any longer) does not elicit a pattern upon amino-acid starvation in vivo. (3) The amino-acid starvation GFP-MAD pattern is abrogated upon depletion of dPARP16, the closest homologue of an established human MARylation enzyme PARP16. Conversely, overexpression of dPARP16 elicits GFP-MAD spot formation (but not YFP-PAD spots, which rule out PARylation events). This is confirmed by the use of a PARylation detection probe YFP-PAD. It allows the visualization of PARylation events consistent with those reported to occur to RNA binding proteins upon arsenate treatment leading to stress granule formation. However, YFP-PAD is not remodelled during amino-acid starvation suggesting that PARylation is not prominent during this stress. Taken together, these evidences demonstrate the specificity of GFP-MAD in binding mono-ADP-ribose and detecting MARylation events (Aguilera-Gomez, 2016).

Second, MAD can be used in cells to follow MARylation in real time. In this regard, GFP-MAD allows the visualisation of an unprecedented display of MARylation events upon amino-acid starvation (but not heat shock or arsenate poisoning) under the form of non-membrane bound, reversible spots/rings in the cytoplasm. This is the first time that MARylation response is visualised in real time during stress (Aguilera-Gomez, 2016).

Third, GFP-MAD has also allowed identification dPARP16 as the enzyme catalysing these events in defined regions of the cytoplasm in a dynamic manner. It is proposed that GFP-MAD spots represent a concentration of MARylated substrates reflecting local dPARP16 activation. This is supported by their co-localization upon expression of both enzyme and probe. This makes GFP-MAD, an efficient activity sensor of the nutrient stress response and dPARP16 the key MARylation enzyme eliciting this response (Aguilera-Gomez, 2016).

Fourth, GFP-MAD can be used for in vitro approaches such as IP and this led to the identification of Sec16 as a MARylation substrate. Last, GFP-MAD and cherry-MAD can be used in vivo and in an anchor-away MARylation assay as an alternative to in vitro approaches using purified components. This has allowed visualization of MARylation in real time and the MARylated region of Sec16 to be mapped (Aguilera-Gomez, 2016).

dPARP16 is necessary and sufficient for Sec body formation upon amino-acid starvation and this creates an unprecedented link between MARylation, metabolic stress and the formation of stress assembly. dPARP16 is necessary for cell survival during amino-acid starvation and recovery. This makes dPARP16 a key enzyme for the cells to specifically cope with amino-acid starvation, as the viability of dPARP16 depleted cells kept in full medium is not compromised. This makes dPARP16 a key survival factor upon amino-acid starvation (Aguilera-Gomez, 2016).

Nutrient starvation in yeast also leads to storage of metabolic enzymes in reversible assemblies (Narayanaswamy, 2009), such as glutamine synthetase or proteasome subunits. Although no PARPs have been identified in Saccharomyces cerevisiae, the regulation of their organisation might be controlled by SIRT, another class of NAD+ dependent protein that also display ADP-ribosylation activity. Conversely, given the abundance of PARPs with predicted MARylation activity in the mammalian genome, it is likely that additional ones, will be required and/or involved in the formation of stress assemblies upon different biological processes, including metabolic stress as described in this study. It has been reported that large Sec bodies did not form in mammalian cells upon conditions used for Drosophila cells, although a remodelling of the early secretory pathway was observed (Zacharogianni, 2014). Therefore, the fine dissection of the signalling pathways involved in Sec body formation will allow recapitulatulation of conditions to trigger their formation in mammalian cells and tissues (Aguilera-Gomez, 2016).

According to RNAseq data of S2 cells in growing conditions and upon amino-acid starvation conditions, dPARP16 has a very low transcriptional level when compared to most genes, suggesting that its protein level is also low. Because dPARP16 moderate overexpression in growing cells leads the detection of MARylation events, it suggests that dPARP16 overexpression leads to its activation. This also suggests that dPARP16 level needs to be kept low in basal conditions to avoid its activation, challenging the detection of its activity in basal conditions. Conversely, dPARP16 is essential during stress, at least amino-acid starvation as depletion of dPARP16 affects the viability of cells during the stress period. How is PARP16 activated upon amino-acid starvation remains to be elucidated. Given the nature of the stress, TORC1 activation would be an ideal pathway, but it has been shown to not be involved in Sec body formation (Zacharogianni, 2014). Another possibility is the fluctuation in the intracellular pH as shown for yeast upon energy deprivation that results in the formation of macromolecular assemblies. However, the time scale is very different (minutes for the drop in pH fluctuation versus hours of starvation for Sec body formation). Furthermore, the assemblies that form upon pH fluctuation do not appear to be mediated by a signalling pathway. This remains to be investigated (Aguilera-Gomez, 2016).

In mammalian cells, PARP16 is activated via auto-MARylation triggered by ER stress (Jwa, 2012). As a result, it MARylates two key kinases of the ER stress response UPR, Ire1 and PERK (Gardner, 2013), leading to their activation (Jwa, 2012) and the unfolded Protein Response. As dPARP16 shares many features with its human counterpart, ER stress could in principle lead to Sec body formation. However, it has been shown previously (Zacharogianni, 2014) that inducing ER stress does not lead to Sec body formation. Although ER stress might be involved in the amino-acid starvation stress response, it is not sufficient to trigger it. This result is reinforced by the demonstration that cell survival is significantly more affected by amino-acid starvation than by ER stress. As a result, dPARP16 appears to be more critical for amino-acid starvation than for ER stress (Aguilera-Gomez, 2016).

This suggests that additional signals are generated during amino-acid starvation. These are under investigation. Another substrate of mammalian PARP16 is karyopherin (Di Paola, 2012), a component required for nuclear export. However, the pharmacological inhibition of nuclear export does not inhibit Sec body formation, suggesting that at least during amino-acid starvation, karyopherin might not play a prominent role and that dPARP16 has different substrates (Aguilera-Gomez, 2016).

One of these substrates is the ERES/Sec body component Sec16 and more specifically a conserved 44 amino-acid sequence (SRDC) in its C-terminus. Indeed, The CAAX version of SRDC recruits cherry MAD to the plasma membrane. Overexpression of SRDC leads to the formation of Sec bodies in a dPARP16 dependent manner, and SRCD rescues Sec body formation in Sec16 depleted cells. This suggests that Sec16-SRDC MARylation is a triggering event in Sec body formation (Aguilera-Gomez, 2016).

The discovery of a short peptide required for the formation of Sec bodies is reminiscent to the existence of an Amyloid Converting Peptide in proteins found in nuclear amyloid bodies in cells upon several stresses. Interestingly, amyloidogenesis is mediated by this motif binding a long non-coding RNA that could be equivalent or comparable to the SRDC MARylation during Sec body formation. Whether the SRCD sequence is also also present in other proteins recruited to Sec bodies remains to be investigated (Aguilera-Gomez, 2016).

In the context of the full-length endogenous protein, SRDC MARylation could act as both a signalling and structural event allowing the recruitment of Sec16 and other ERES components into Sec bodies. However, SRCD on its own is not recruited to Sec bodies, and it may act only as a signalling event for Sec body formation. The nature of this signalling needs to be further investigated. This is in line with the fact that GFP-MAD is not readily observed within the Sec body core. GFP-MAD appears as a ring/spot at the base of Sec bodies. Although this is consistent with the protein packing and competing binding that takes place during the formation of stress assemblies (that most likely would exclude GFP-MAD), it might also suggest that MARylated Sec16 forms a signalling platform. This would lead to the modifications of other Sec body components allowing their incorporation (Aguilera-Gomez, 2016).

Interestingly, PARylation has been proposed to preferentially occur on LCSs (Low complexity sequences, that is, region of poor amino-acid diversity). These are normally thought to correspond to disordered regions. Sec16 is rich in LCSs (Zacharogianni, 2014) and SRDC is intrinsically disordered, therefore accessible to be modified by dPARP16. Taken together, it is postulated that Sec16 is a stress response protein and a new substrate for the pro-survival dPARP16 upon amino-acid starvation (Aguilera-Gomez, 2016).

Neutral competition for Drosophila follicle and cyst stem cell niches requires vesicle trafficking genes

The process of selecting for cellular fitness through competition plays a critical role in both development and disease. The germarium, a structure at the tip of the ovariole of a Drosophila ovary, contains two follicle stem cells (FSCs) that undergo neutral competition for the stem cell niche. Using the FSCs as a model, a genetic screen through a collection of 126 mutants in essential genes on the X chromosome was performed to identify candidates that increase or decrease competition for the FSC niche. Approximately 55% and 6% of the mutations screened were obtained as putative FSC hypo- or hypercompetitors, respectively. A large majority of mutations were found in vesicle trafficking genes (11 out of the 13 in the collection of mutants) are candidate hypocompetition alleles, and the hypocompetition phenotype was confirmed for four of these alleles. Sec16 and another COP II vesicle trafficking component, Sar1, are required for follicle cell differentiation. Lastly, it was demonstrated that although some components of vesicle trafficking are also required for neutral competition in the cyst stem cells (CySCs) of the testis, there are important tissue-specific differences. These results demonstrate a critical role for vesicle trafficking in stem cell niche competition and differentiation, and a number of putative candidates for further exploration were identified (Cook, 2017).

The follicle stem cells in the Drosophila ovary are a highly tractable model of stem cell niche competition. The Drosophila ovary is comprised of long strands of developing follicles, called ovarioles, and a pair FSCs resides at the anterior tip of each ovariole in a structure called the germarium. These FSCs divide during adulthood to provide the follicle cells that surround germ cell cysts during follicle formation. FSCs are regularly lost and replaced during adulthood, and several studies have identified genes that increase the rate of FSC loss. In most cases, the mutations investigated in these studies disrupt the ability of the mutant FSC to adhere to the niche or transduce niche signals and thus are presumed to cause the mutant stem cell to be lost in a cell-autonomous manner. However, the suggestion that stem cells may compete with the daughters of neighboring stem cells for niche occupancy raises the possibility that a mutation in a competing mutant lineage could act in a noncell-autonomous manner to influence the likelihood that a neighboring wild-type lineage will be lost and replaced (Cook, 2017).

This study has generated a new resource for investigating the genetic basis of stem cell niche competition. The collection of confirmed and candidate competition mutations demonstrates the breadth of cell functions that influence niche competition, and will allow for further study into many different facets of this process. One hyper-competition mutation and seven additional candidate hyper-competition mutations were generated in genes involved in a variety of functions, including mitochondrial function (sicily, tumor suppression (Rbf), and protein ubiquitination (bendless). The diversity of this list of candidates suggests that effects on many different cellular processes can lead to a hyper-competition phenotype. Yet the vast majority of mutations that were studied do not cause hyper-competition, suggesting that the ultimate cause(s) of hyper-competition are more constrained, and that the mechanism of hyper-competition may be similar in these diverse mutants (Cook, 2017).

A previously unstudied aspect of niche competition revealed by this screen is the involvement of vesicle trafficking genes. The Sec16A, shiA, wusA, and CragA mutations that that were examined in this study represent a broad range of vesicle trafficking functions. Specifically, Sec16 is a central regulator of exocytosis, whereas wurst and shibire function primarily during endocytosis, and Crag is required in follicle cells for trafficking of basement membrane components to the basal surface. The shiA (K10X) and wusA (Q307X) alleles, which produced the most severe effects on DE-cad localization, contain nonsense mutations and are most likely to be amorphic alleles; while CragA (C1372S) and Sec16A (P1926S) contain missense mutations and are more likely to be hypomorphic alleles. Comparison of the relatively mild phenotypes caused by homozygosity for Sec16A to the more severe phenotypes caused by RNAi knockdown of Sec16A further support the conclusion that Sec16A is a hypomorphic allele. Interestingly, despite these differences, all four mutations caused strong hypo-competition phenotypes in the FSC lineage. This suggests that the process of niche competition is very sensitive to the function of these genes and is able to efficiently eliminate stem cell lineages with even only mild defects in vesicle trafficking (Cook, 2017).

The finding of this study suggest at least two possible reasons why impaired vesicle trafficking causes hypo-competition. First, DE-cad is known to be required for FSC maintenance, so the disruption of DE-cad localization to the membrane in vesicle trafficking mutants could at least partially account for the hypo-competition phenotype. Second, vesicle trafficking is required for functional EGFR signaling in MDCK cells, and this study found decreased pERK levels in the vesicle trafficking mutant clones. Since EGFR signaling is also essential for FSC self-renewal, the reduction in EGFR signaling may be another factor that contributes to the hypo-competition phenotype. However, EGFR signaling is normally downregulated as cells exit the FSC niche region, so it is unclear whether the decreased pERK levels in these mutant clones are a cause of the hyper-competition phenotype or a consequence of reduced association with the niche (for example, because DE-cad levels on the membrane are low). In addition, it is also unclear why the loss of detectable pERK in these mutant clones is not associated with a cell polarity defect, as has been observed in EGFR null clones. It may be that the vesicle trafficking alleles investigated in this study reduce the levels of EGFR signaling to a level that is below the limit of detection but sufficient to maintain cell polarity; or that the vesicle trafficking mutants impair the branch of EGFR signaling leading to ERK phosphorylation, but not the branch going through LKB1 and AMPK that is important for the maintenance of cell polarity in the FSC lineage. Alternately, it could be that the decrease in EGFR signaling is more gradual in the vesicle trafficking mutant clones than it is in EGFR mutant clones, so the vesicle trafficking mutant clones are able to grow normally for some time before EGFR signaling decreases to the point at which it is both undetectable by pERK staining and unable to promote FSC self-renewal or follicle cell polarity. Additional studies of these and other FSC niche competition mutants identified here will be important to clarify these issues (Cook, 2017).

In the testis, knockdown of Sec16 also affected CySC niche competition rather than self-renewal or differentiation, whereas knockdown of shi likely impaired CySC self-renewal or survival. Interestingly, knockdown of Crag had no effect on CySC retention in the niche but caused an unusual differentiation defect in which mutant cells failed to move out of the niche region. These differences indicate that there are tissue-specific aspects to the process of self-renewal and niche competition, and provide additional evidence that the hypo-competition phenotypes observed in the FSC niche are not due to a generic defect that would affect all cells equally. Overall, these studies demonstrate that mutations in multiple types of vesicle trafficking genes cause hypo-competition in both the ovary and testis. Vesicle trafficking is essential for a diverse array of cellular functions, and thus may function as a node, integrating outputs from these different cellular functions into a readout that influences the overall fitness of the cell for occupying the niche. Further investigation will make it possible to organize these and other mutations that cause niche competition phenotypes into common pathways and, ultimately, to understand whether and how stem cell niche competition promotes the maintenance of a healthy population of stem cells in each tissue throughout adulthood (Cook, 2017).

Loss of a Clueless-dGRASP complex results in ER stress and blocks integrin exit from the perinuclear endoplasmic reticulum in Drosophila larval muscle

Drosophila Clueless (Clu) and its conserved orthologs are known for their role in the prevention of mitochondrial clustering. This study uncovered a new role for Clu in the delivery of integrin subunits in muscle tissue. In clu mutants, αPS2 integrin (Inflated), but not βPS integrin, abnormally accumulates in a perinuclear endoplasmic reticulum (ER) subdomain, a site that mirrors the endogenous localization of Clu. Loss of components essential for mitochondrial distribution do not phenocopy the clu mutant alphaPS2 phenotype. Conversely, RNAi knockdown of the Drosophila Golgi reassembly and stacking protein GRASP55/65 recapitulates clu defects, including the abnormal accumulation of αPS2 and larval locomotor activity. Both Clu and dGRASP proteins physically interact and loss of Clu displaces dGRASP from ER exit sites, suggesting that Clu cooperates with dGRASP for the exit of alphaPS2 from a perinuclear subdomain in the ER. This study also found that Clu and dGRASP loss of function leads to ER stress and that the stability of the ER exit site protein Sec16 is severely compromised in the clu mutants, thus explaining the ER accumulation of αPS2. Remarkably, exposure of clu RNAi larvae to chemical chaperones restores both αPS2 delivery and functional ER exit sites. It is proposed that Clu together with dGRASP prevents ER stress and therefore maintains Sec16 stability essential for the functional organization of perinuclear early secretory pathway. This, in turn, is essential for integrin subunit αPS2 ER exit in Drosophila larval myofibers (Wang, 2015).

The data demonstrate a novel role for Clu in αPS2 exit from the perinuclear ER in larval muscle that is different from previously reported roles. The first established function is in the prevention of mitochondrial clustering. The second role of Clu regulates aPKC activity in neuroblast stem cell divisions (Goh, 2013). A third role for Clu was published just before submission of this manuscript. Mammalian CLUH can function as an mRNA-binding protein for RNAs encoding nuclear mitochondrial proteins, thus possibly providing a link for mitochondrial biogenesis and localization (Gao, 2014). Thus, Clu is a multifaceted protein whose cellular and developmental roles are just beginning to be elucidated (Wang, 2015).

This study shows that αPS2 is synthesized from a pool of mRNA that is targeted around the nucleus. As αPS2 is a transmembrane protein, this would allow for local synthesis of this protein in the perinuclear ER. This same idea has been proposed in polarized cells, where the coupling of mRNA retention and local translational allows for efficient sorting to the final sites of membrane deposition and/or secretion. When the machinery for αPS2 ER exit is disrupted, αPS2 is retained in the perinuclear ER, as observed in Clu and dGRASP. How αPS2 mRNA is targeted to this location is not known. The ER can form either networked tubules or stacked sheets, the latter being more abundant around nuclei and it is therefore possible that ER structure plays a role in mRNA targeting (Wang, 2015).

Both Clu and dGRASP form a complex that functionally localizes to ER exit sites (ERES). The role of this complex could be either direct, such as an interaction with ER cargo receptors such as p24 family members, or indirect. For instance, loss of Clu or dGRASP could affect the microtubule (MT) network and compromise the functional integrity of ERES. Previous data shows that the MT cytoskeleton is closely associated with the reorganization of 'transitional ER' tER-Golgi units near the nuclear envelope in rat contractile myofibers. However, this study ruled out a role for the MT cytoskeleton in αPS2 delivery. Loss of Clu or dGRASP did not alter the organization of the MT network in larval muscle cells. Furthermore, disruption of the MT cytoskeleton by muscle-specific overexpression of the MT-severing protein Spastin in L3 larval muscles did not recapitulate the perinuclear accumulation of αPS2 (Wang, 2015).

Clu acts to mediate αPS2 export through modulation of Sec16 stability, a key factor required for COPII coated vesicle dynamics. This study shows that Clu and dGRASP act to inhibit ER stress. Upon loss of Clu, ER stress increases, leading to Sec16 degradation and impairment of αPS2 export, and ER retention. Importantly, alleviating ER stress with the chemical chaperones TUDCA and 4PBA suppressed both αPS2 accumulation and the size of ERES. This data provides at least one mechanism for the regulation of αPS2 transport by Clu-dGRASP in myofibers (Wang, 2015).

The biological inputs that trigger ER stress in muscle tissue are not clear. Studies in Drosophila follicle cells support the intriguing hypothesis that integrins trigger their own mode of transport in response to mechanical stress. The physical tension generated during epithelial remodeling induces an upregulation of dgrasp mRNA and is dependent upon integrins and the subsequent recruitment and/or activation of RhoA and the LIM protein PINCH. Interestingly, elevated PINCH levels also suppress hypercontraction muscle mutants. Thus, maybe PINCH is a key sensory component in tissues that sense, transduce, and alter secretion routes of proteins to withstand changes in physical forces. Supporting this idea are multiple pieces of evidence where changes in patterned muscle activity alter the distribution of the Golgi and ERES. Furthermore, The RNA binding protein HOW is involved in dgrasp mRNA stability the in the follicular epithelium and interesting, how mutants show a muscle phenotype . If Clu is acting as a sensor in transducing mechanical stress, for example, it may have the ability to alter the trafficking of proteins in response to such physiological changes (Wang, 2015).

The general organization of ERES and the Golgi complex seem conserved between Drosophila and mammalian skeletal muscles, where these organelles are broadly distributed throughout the cell with accumulation around nuclei. Studies of glycoprotein processing show that multiple delivery routes exist in multinucleated myotubes. For example, influenza virus hemagglutinin (HA) is transported through the Golgi to the cell surface in rat L6 muscle cells. However, half of the pool of labeled vesicular stomatitis virus (VSV) G protein exits the ER but gets shuttled into intracellular vesicles independent of the Golgi. It is not surprising that the complexity of muscle cells may require multiple or redundant routes for membrane delivery (Wang, 2015).

Like αPS2 in this system, the α integrin subunit (αPS1) in the Drosophila follicular epithelium is also retained in the ER in the absence of dGRASP function and reaches the plasma membrane in a Golgi independent manner. This leads to the question as to whether αPS2 in larval muscles also bypasses the Golgi. Preliminary results of Syntaxin 5 (an essential SNAREs for protein transport to and through the Golgi) knockdown showed severely impaired larval survival, but did not phenocopy the clu or dgrasp αPS2 accumulation phenotype. This suggests that αPS2 could bypass the Golgi. However, biochemical evidence demonstrating the presence or absence of Golgi-specific post translational modifications have proven difficult to gather and it remains an open question. Interestingly, in HeLa cells, Golgi bypass of CFTR has been linked to ER stress leading to GRASP55 binding to the C-terminal PDZ1 domain of CFTR (Wang, 2015).

One outcome from this work is a departure from the notion that α/β heterodimer formation is a prerequisite for ER exit, and therefore the accumulation of αPS2, but not βPS is counterintuitive. Of note, βPS is not excluded from the perinuclear ER, so the role of Clu as a chaperone might still hold true. Nevertheless the ER export of integrins (as a complex or as individual subunits), at least in Drosophila, might be more complex than anticipated and might change at different stages of development. Taken together, require more studies to determine what domains of Clu and/or interacting partners are essential for various cellular activities (Wang, 2015).

A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation

Nutritional restriction leads to protein translation attenuation that results in the storage and degradation of free mRNAs in cytoplasmic assemblies. This study shows in Drosophila S2 cells that amino-acid starvation also leads to the inhibition of another major anabolic pathway, the protein transport through the secretory pathway, and to the formation of a novel reversible non-membrane bound stress assembly, the Sec body that incorporates components of the ER exit sites. Sec body formation does not depend on membrane traffic in the early secretory pathway, yet requires both Sec23 and Sec24AB. Sec bodies have liquid droplet-like properties, and they act as a protective reservoir for ERES components to rebuild a functional secretory pathway after re-addition of amino-acids acting as a part of a survival mechanism. Taken together, it is proposed that the formation of these structures is a novel stress response mechanism to provide cell viability during and after nutrient stress (Zacharogianni, 2014).

This study describes a novel, reversible, and non-membrane bound structure, the Sec body that forms in response to nutrient stress. Sec bodies comprise proteins that in normal growth conditions function as endoplasmic reticulum exit site (ERES) components, including subunits of the COPII coat, namely Sec23, Sec24AB, Sec24CD, and Sec31 as well as Sec16, the upstream ERES organizer. A noticeable exception is the small GTPase Sar1. One reason for this could be that Sar1 is devoid of low complexity sequences (LCS) and this is currently under further investigation (Zacharogianni, 2014).

Interestingly, the components of the other coats were seemingly not incorporated into Sec bodies but also did not form other structures, suggesting that remodeling of the ERES is sufficient to ensure inhibition of protein transport through the secretory pathway. In this regard, Sec body formation constitutes also a novel mechanism for attenuation/inhibition of protein transport through the secretory pathway. Cells can disperse ERES and Golgi components into the cytoplasm, as reported during mitosis the Golgi is fragmented (Zacharogianni, 2014).

The dramatic remodeling of the ERES is described in this study appears to be specific for amino-acid starvation, possibly underlining the acute and severe nature of this particular stress. It is also different from the response to serum starvation that requires ERK7 (Zacharogianni, 2011). ERK7 appears to be involved to a small extent in the amino-acid starvation response, possibly facilitating the initial dispersion of a fraction of the ERES into the cytoplasm, as when depleted, it prevents Sec body formation upon amino-acid starvation by about 20% (Zacharogianni, 2011). However, other signaling pathways, yet unidentified, are clearly at stake (Zacharogianni, 2014).

Interestingly, one of Sec body components is Sec16, a protein with a localization that is also modulated upon serum starvation. Furthermore, Sec16 is also phosphorylated in response to EGF signaling in human cells. It could therefore be emerging as one of platform integrating nutrient and growth factors availability (Zacharogianni, 2014).

Sec bodies are novel stress structures and this study has shown that they are not autophagosomes (or substrates of autophagy), not lipid droplets and not CUPS as they are devoid of dGRASP that is found quantitatively re-distributed in the cytoplasm upon amino-acid starvation, suggesting a modification involving its lipid anchor or modification of its N-terminus. Sec bodies are also different from large reversible structures containing COPII components that have been described in yeast in a number of specific COPII mutants Sec12-4 and Sec16-2. These structures are thought to result from an imbalance between cargo incorporation in COPII-coated vesicles and the coat formation, and lowering the cargo load by inhibiting protein translation prevented their appearance. Sec body formation is, however, insensitive to translation inhibition by cycloheximide and is therefore different from these yeast structures. Last, it was also shown that Sec bodies are distinct from Stress Granules and P-bodies that also form upon amino-acid starvation. Therefore, Sec bodies are novel structures (Zacharogianni, 2014).

Formation of mesoscale protein assemblies like Stress Granules, P-bodies or, more recently, Sec bodies is emerging as a general response to stress and especially nutrient stress, and is gaining increasing attention. For instance, in yeast under nutrient limiting conditions, metabolic enzymes and stress response proteins form reversible foci, such as purinosomes containing enzymes of the purine biosynthetic pathway, proteasome storage granules upon glucose restriction, or, as recently described, glutamine synthetase filaments (Zacharogianni, 2014).

In challenging conditions, areas of localized biochemistry in the cytoplasm can be advantageous, as reagents and possibly energy can be focused to these specific areas. The reorganization of the cytoplasm through non-membrane bound protein assemblies could confer this rapid and spatio-temporally defined compartmentalization. In this regard, this study has found that Sec bodies confer a fitness advantage to the cells under starvation. However, some stress assemblies (especially cytoplasmic RNP granules) can form dysfunctional RNA-protein assemblies that become irreversible and toxic for the cell. For instance, Stress Granule components have a strong relationship with degenerative diseases, such as ALS and laminopathies. Whether Sec body components could also form such deleterious aggregates remains to be established (Zacharogianni, 2014).

Some of these mesoscale assemblies have liquid-like properties. These so-called liquid droplets are generally spherical and dynamic and form via phase separation (liquid demixing) of their components from the cytoplasm like a drop of oil in water. Their components display different rates of diffusion within the assembly and in the surrounding cytoplasm. They form via transient and weak protein-protein and protein-RNA interactions mediated by low amino-acid diversity stretches (low complexity sequences, LCS), prone to engage in such interactions. Stress granules and P-bodies have been shown to be liquid droplets, and this study shows that Sec bodies exhibit clear liquid droplet features as underlined by their spherical morphology, their reversibility, FRAP properties, and LCS content (Zacharogianni, 2014).

In this regard, the presence of LCSs both in Sec16 and Sec24 is intriguing considering their role in cells under normal growth conditions where they act in sequence with many others to form the COPII coat. How the LCSs are shielded to make proteins competent for their function in COPII coat formation in growing cells remains to be investigated but the interaction with both cargo and Sec23 might be instrumental to their functioning as coat subunits. This study shows that the LCS rich domain of Sec24AB is sufficient and necessary for Sec body incorporation upon amino-acid starvation, but not sufficient to induce Sec body formation. This has similarity to Tia1, a key protein necessary for Stress Granule formation that has an LCS-prion like domain that is necessary to form stress granules (Zacharogianni, 2014).

As mentioned above, the structures that fall in the liquid droplet category have been described to form through weak protein-RNA interactions. Although Sec bodies do not appear to contain RNAs, it is proposed that they are nonetheless liquid droplets. The absence of RNA might account for the very low and slow recovery observed after complete photobleaching of whole Sec bodies when compared to Stress Granules that recovers to a higher degree. Shuttling of mRNA in and out of Stress Granules could drive more exchange between the structure and the surrounding cytoplasm, and this probably does not occur in Sec bodies. However, instead of protein-RNA interactions, Sec body components could establish weak protein-protein interactions helped by molecular modifications that could trigger conformational changes and perhaps exposure of their LCS (Zacharogianni, 2014).

Importantly, one of the key features of a liquid droplet is to be efficiently reversible, and this study shows that Sec bodies are rapidly and completely reversible upon stress relief. This shows that they act as a reservoir of the ERES components that can be quickly remobilized to re-build a functional organelle, so that protein transport through the secretory pathway can resume once stress is relieved in order to support cell proliferation. Furthermore, Sec bodies have a role in protecting ERES components from degradation during starvation. That strengthens the fact that Sec bodies are neither autophagosomes nor a substrate of autophagy, unlike Stress Granules, which are reported to be cleared by autophagy. Last, this study showed that Sec body formation is critical for the cell viability during amino-acid starvation. It suggests a pro-survival mechanism, perhaps through the recruitment and inactivation of pro-apoptotic factors. This remains to be investigated (Zacharogianni, 2014).

Taken together, amino-acid starvation inhibits both protein translation and protein transport through the secretory pathway and, similarly for both processes, results in the concomitant formation of cytoplasmic stress assemblies where key components necessary for cell survival are stored: untranslated mRNAs in Stress Granules and ERES components in Sec bodies (Zacharogianni, 2014).

ERK7 is a negative regulator of protein secretion in response to amino-acid starvation by modulating Sec16 membrane association

RNAi screening for kinases regulating the functional organization of the early secretory pathway in Drosophila S2 cells has identified the atypical Mitotic-Associated Protein Kinase (MAPK) Extracellularly regulated kinase 7 (ERK7) as a new modulator. ERK7 was found to negatively regulate secretion in response to serum and amino-acid starvation, in both Drosophila and human cells. Under these conditions, ERK7 turnover through the proteasome is inhibited, and the resulting higher levels of this kinase lead to a modification in a site within the C-terminus of Sec16, a key ER exit site component. This post-translational modification elicits the cytoplasmic dispersion of Sec16 and the consequent disassembly of the ER exit sites, which in turn results in protein secretion inhibition. ER exit site disassembly upon starvation was found to be TOR complex 1 (TORC1) independent, showing that under nutrient stress conditions, cell growth is not only inhibited at the transcriptional and translational levels, but also independently at the level of secretion by inhibiting the membrane flow through the early secretory pathway. These results reveal the existence of new signalling circuits participating in the complex regulation of cell growth (Zacharogianni, 2011).

Over the past three decades genetic, biochemical and morphological analyses have advanced understanding of the molecular machineries mediating the functional organization of the early secretory pathway. Key factors have been identified to have a role in the ER to Golgi trafficking and the architecture of this pathway. In particular, Sec16 has been identified as a large hydrophilic protein that is tightly associated with the cup-shaped ER structures that characterize the ER exit sites, where it sustains its function in the recruitment of COPII components to increase vesicular budding and transport (Connerly, 2005; Bhattacharyya, 2007; Ivan, 2008; Hughes, 2009). To gain insight on how signalling molecules regulate the organization of the early secretory pathway, 245 Drosophila kinases were depleted by RNAi and their effect on tER-Golgi units was assessed by a microscopy-based screen (Zacharogianni, 2011).

About 10% of the depleted kinases (26 out of 245) were confirmed to alter the organization of tER-Golgi units. Most of the depletions led to an increase in their number (that is referred to as MG phenotype). A clustering of the tER-Golgi units was sometimes observed but not their complete disassembly, similar to the results obtained in the screen for ER proteins. In addition, for a number of kinases, an increase was also observed in the cell sizenthat remains unexplained by the parameters tested (cell-cycle inhibition, anterograde transport, TORC1 activation and lipid droplet biogenesis) in this study (Zacharogianni, 2011).

Several kinases were identified both in the current screen and a recently published similar screen performed in human cells. The overlap between the two screens is small. Although technical differences in the screening methods, cell-type specificity and genetic redundancy could explain this paucity, this might also suggest that despite the functional organization of the early secretory pathway mediated by a relatively conserved set of factors, its regulation by signalling molecules may vary from organism to organism (Zacharogianni, 2011).

The screen identified ERK7 that encodes a protein homologous to mammalian ERK7/8 also known as MAPK15. Interestingly, the screen in HeLa cells did not pick MAPK15 because the phenotype is only obvious upon starvation, not in normal growth conditions. When depleted from S2 cells, ERK7 led to an MG phenotype and when overexpressed, it downregulated secretion. As secretion is a key factor in cell growth, it suggests that ERK7 has an inhibitory role on cell growth. In support of this, ERK7 expression is very low (and even downregulated) during larval stages that are characterized by massive growth. Growth inhibition by ERK7 has also been observed for mammalian ERK7/8. For instance, overexpression of MAPK15 has been found to negatively regulate hippocampal neurite growth. As neurite growth is linked to ER to Golgi trafficking in Drosophila and in mammals, the neurite growth phenotype could be linked to disassembly of ER exit sites. In addition, a decreased expression of ERK8 has been reported in breast cancer cell lines, indicating that this kinase could function as tumour suppressor by reducing cell growth. Overexpressing ERK7 and ERK8 also inhibits DNA synthesis in COS cells and HEK cell proliferation, although the mechanism underlying growth inhibition is clearly different. Drosophila ERK7 has not been genetically characterized during development, but it has been identified in screens searching for regulators of cell cycle and calcium signalling (Zacharogianni, 2011).

Strikingly, ERK7 overexpression has a very similar effect on Sec16 (and Sec23) to that of serum and amino-acid starvation. Furthermore, lowering ERK7 activity partially rescues the tER site disassembly phenotype induced by serum and amino-acid starvation, in both S2 and HeLa cells, underlying the central role of ERK7 in this event. Moreover, this study has shown that ERK7 is stabilized upon amino-acid deprivation, which protects it from degradation through the proteasome. Interestingly, ERK7 harbours two βTRCP phospho-degrons. βTRCP is known to interact with SCF and the E3 ligase Cullin, to promote the phosphorylation-dependent ubiquitination and proteasomal degradation of its targets. As mammalian ERK7 is one of the targets, it is hypothesized that amino-acid starvation could stabilize ERK7 by preventing its targeting by the SCFβTRCP. This will need to be further investigated (Zacharogianni, 2011).

However, inhibiting endogenous ERK7 degradation by treating S2 cells cultured in full medium with MG132 did not lead to Sec16 dispersion. This could be due to a number of reasons. One is that MG132 inhibits the proteasome without preventing the ubiquitination of the targets, and ERK7 ubiquitination could possibly compromise its kinase activity. The second is that starvation could also result in the upregulation of a factor needed for ERK7 localization to ER exit sites so that it can modify Sec16. Third, amino-acid starvation might not only lead to ERK7 stabilization, but also to its activation. All this, of course, could be overridden by ERK7 overexpression, as it leads to the dispersion of Sec16 even in the presence of nutrient and growth factors. Last, amino-acid starvation is likely to activate parallel pathways and this would explain why lowering ERK7 only partially rescues the induced Sec16 dispersion/aggregation phenotype. In this regard, it should be noted that serum and amino-acid starvation of HeLa cells results in a milder ER exit site disassembly but is completely rescued by MAPK15 depletion (Zacharogianni, 2011).

Serum starvation-induced tER site disassembly is not inhibited by MEK inhibitors, nor is it by PKC and p38 inhibitors; neither does it involve pre-translational events, as blocking translation with cycloheximide addition while serum starving the cells exacerbates the starvation effect. More surprisingly, although amino-acid starvation is well known to inhibit TORC1 in many organisms, resulting in growth inhibition through decreased protein synthesis and increased protein degradation (through stimulation of autophagy). TORC1 does not seem to be involved in the cessation of secretion through the tER disassembly, as shown by the lack of effect of rapamycin, and Raptor depletion or the addition of insulin. It is, therefore, argued that although secretion is clearly a mechanism controlling cell growth in response to nutrients, the cell uses a signalling pathway involving ERK7, but distinct from TORC1, to downregulate protein transport upon starvation. Taken together, it means that nutrient starvation controls cell growth not only through translation inhibition and downregulation of biosynthetic pathway as documented by others, but also through secretion inhibition by using a novel signalling pathway that has been identified in this study. Interestingly, secretion rate has also been shown to be inhibited by 50% in yeast upon amino-acid starvation and yeast could prove a useful model organism to dissect the signalling cascade involved in transducing nutrient restriction (Zacharogianni, 2011).

ERK7 exerts its inhibitory role on secretion through the Sec16 release from tER sites, in a kinase-dependent manner, leading to their disassembly. Since the possibility that ERK7 modifies the putative Sec16 receptor at the tER sites is ruled out, Sec16 itself is likely the target of the ERK7 signalling pathway. It could be indirect with other kinases downstream of ERK7, such as CK2 but this has been ruled out. Furthermore, despite the large battery of inhibitors used in this study, none have inhibited serum starvation triggered Sec16 dispersion downstream of ERK7. This, therefore, opens the possibility that Sec16 is a direct ERK7 substrate. One serine/threonine residue situated in the 'starvation responsive domain' (aa 1740-1880) could be phosphorylated by ERK7. Alternatively, this domain could act as a landing platform for ERK7 that could in turn phosphorylate residues situated in other domains of Sec16. A more extensive proteomics and mutation analysis will be required to fully elucidate this issue (Zacharogianni, 2011).

In the same vein, the Sec16 responsive domain may act as a landing platform for a phosphatase. Drosophila Sec16 harbours many phosphorylation sitesthat are mostly located in the minimal domain for tER site localization. This putative phosphatase could specifically dephosphorylate specific residues of the localization domain, resulting in the Sec16 release. However, incubation with the broad-spectrum protein phosphatase inhibitors (sodium orthovanadate, inhibitor of protein phosphor-tyrosine and alkaline phosphatases and okadaic acid, inhibitor of phospho-serine/threonine protein phosphatases 1 and 2) did not protect against the tER disassembly induced by serum starvation (Zacharogianni, 2011).

Although this work points to Sec16 as an ERK7 target (whether direct or not) and to tER site maintenance as a way to control secretion, other key proteins of the secretory pathway might also be targets, leading to an inhibition of secretion at multiple levels within the secretory pathway. This will need further research (Zacharogianni, 2011).

The results presented here also strengthen the relationship between the secretory pathway and signalling, and in particular the role of ERKs on ER exit sites. In HeLa cells, ERK2 was identified to directly phosphorylate human Sec16 on Threonine 415 (T415) both in vivo and in vitro followed by its recruitment to ERESs, increased ERES number and anterograde ER to Golgi trafficking. Although ERK2 is clearly important in human cells, the results show that it does not seem to have a role in S2 cells. This suggests that in S2 cells, inhibiting secretion when serum and/or amino acids are missing is not only a passive mechanism of not stimulating ERK2, but an active mechanism involving ERK7. Furthermore, HeLa cells also exhibit this active mechanism (Zacharogianni, 2011).

In short, ERK2 has an opposite effect on Sec16 from the proposed function for ERK7/MAPK15: (1) Growth factors stimulate Ras and ERK2 that directly phosphorylate Sec16 on T415. (2) This results in an increased mobility of Sec16 (Sec16 recruitment to ERES is increased, either from the cytosol or from the general ER membrane). (3) The number of ERES as well as the secretory capacity increase. Conversely, (1') Amino-acid starvation stabilizes ERK7 in a TORC1-independent pathway. This induces Sec16 phosphorylation in a 'starvation responsive domain'-dependent manner. (2') This results in Sec16 release from the tER sites leading to (3') tER site disassembly and ER-plasma membrane transport inhibition, thus negatively regulating cell growth (Zacharogianni, 2011).

Taken together, these results point towards ERK7 as a novel mediator of nutrient availability in the control of secretion and provide a framework for a better mechanistic understanding of the regulation of protein secretion and cell growth as a response to environmental stimuli (Zacharogianni, 2011).

Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif

tER sites are specialized cup-shaped ER subdomains characterized by the focused budding of COPII vesicles. Sec16 has been proposed to be involved in the biogenesis of tER sites by binding to COPII coat components and clustering nascent-coated vesicles. This study shows that Drosophila Sec16 (dSec16) acts instead as a tER scaffold upstream of the COPII machinery, including Sar1. dSec16 is required for Sar1-GTP concentration to the tER sites where it recruits in turn the components of the COPII machinery to initiate coat assembly. Last, this study shows that the dSec16 domain required for its localization maps to an arginine-rich motif located in a nonconserved region. A model is proposed in which dSec16 binds ER cups via its arginine-rich domain, interacts with Sar1-GTP that is generated on ER membrane by Sec12 and concentrates it in the ER cups where it initiates the formation of COPII vesicles, thus acting as a tER scaffold (Ivan, 2008).


Functions of Sec16 orthologs in other species

Sec16A is critical for both conventional and unconventional secretion of CFTR

CFTR is a transmembrane protein that reaches the cell surface via the conventional Golgi mediated secretion pathway. Interestingly, ER-to-Golgi blockade or ER stress induces alternative GRASP-mediated, Golgi-bypassing unconventional trafficking of wild-type CFTR and the disease-causing DeltaF508-CFTR, which has folding and trafficking defects. This study shows that Sec16A, the key regulator of conventional ER-to-Golgi transport, plays a critical role in the ER exit of protein cargos during unconventional secretion. In an initial gene silencing screen, Sec16A knockdown abolished the unconventional secretion of wild-type and DeltaF508-CFTR induced by ER-to-Golgi blockade, whereas the knockdown of other COPII-related components did not. Notably, during unconventional secretion, Sec16A was redistributed to cell periphery and associated with GRASP55 in mammalian cells. Molecular and morphological analyses revealed that IRE1alpha-mediated signaling is an upstream regulator of Sec16A during ER-to-Golgi blockade or ER stress associated unconventional secretion. These findings highlight a novel function of Sec16A as an essential mediator of ER stress-associated unconventional secretion (Piao, 2017).

Microscopy analysis of reconstituted COPII coat polymerization and Sec16 dynamics

The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. However, it remains unclear how these molecules drive COPII coat polymerization. This study characterized the spatiotemporal relationships between the Saccharomyces cerevisiae COPII components during their polymerization by performing fluorescence microscopy of an artificial planar membrane. Sar1 was shown to dissociate from the membrane shortly after the COPII coat recruitment, and Sar1 is then no longer required for the COPII coat to bind to the membrane. Furthermore, Sec16 was found to be incorporated within the COPII-cargo clusters, and this was found to be dependent on the Sar1 GTPase cycle. These data show how Sar1 drives the polymerization of COPII coat and how Sec16 is spatially distributed during COPII coat polymerization (Iwasaki, 2017).

TANGO1 recruits Sec16 to coordinately organize ER exit sites for efficient secretion

Mammalian endoplasmic reticulum (ER) exit sites export a variety of cargo molecules including oversized cargoes such as collagens. However, the mechanisms of their assembly and organization are not fully understood. TANGO1L is characterized as a collagen receptor, but the function of TANGO1S remains to be investigated. This study shows that direct interaction between both isoforms of TANGO1 and Sec16 is not only important for their correct localization but also critical for the organization of ER exit sites. The depletion of TANGO1 disassembles COPII components as well as membrane-bound ER-resident complexes, resulting in fewer functional ER exit sites and delayed secretion. The ectopically expressed TANGO1 C-terminal domain responsible for Sec16 binding in mitochondria is capable of recruiting Sec16 and other COPII components. Moreover, TANGO1 recruits membrane-bound macromolecular complexes consisting of cTAGE5 and Sec12 to the ER exit sites. These data suggest that mammalian ER exit sites are organized by TANGO1 acting as a scaffold, in cooperation with Sec16 for efficient secretion (Maeda, 2017).

Sec16 alternative splicing dynamically controls COPII transport efficiency

The transport of secretory proteins from the endoplasmic reticulum (ER) to the Golgi depends on COPII-coated vesicles. While the basic principles of the COPII machinery have been identified, it remains largely unknown how COPII transport is regulated to accommodate tissue- or activation-specific differences in cargo load and identity. This study shows that activation-induced alternative splicing of Sec16 controls adaptation of COPII transport to increased secretory cargo upon T-cell activation. Using splice-site blocking morpholinos and CRISPR/Cas9-mediated genome engineering, this study showed that the number of ER exit sites, COPII dynamics and transport efficiency depend on Sec16 alternative splicing. As the mechanistic basis, it is suggested that the C-terminal Sec16 domain is a splicing-controlled protein interaction platform, with individual isoforms showing differential abilities to recruit COPII components. This work connects the COPII pathway with alternative splicing, adding a new regulatory layer to protein secretion and its adaptation to changing cellular environments (Wilhelmi, 2016).

Regulation of Sec16 levels and dynamics links proliferation and secretion

A broader mechanistic understanding of the integration of the early secretory pathway with other homeostatic processes such as cell growth is currently lacking. This study explored the possibility that Sec16A, a major constituent of endoplasmic reticulum exit sites (ERES), acts as an integrator of growth factor signaling. Surprisingly, Sec16A was found to be a short-lived protein that is regulated by growth factors in a manner dependent on Egr family transcription factors. It was hypothesized that Sec16A acts as a central node in a coherent feed-forward loop that detects persistent growth factor stimuli to increase ERES number. Consistent with this notion, Sec16A is also regulated by short-term growth factor treatment that leads to increased turnover of Sec16A at ERES. Finally, Sec16A depletion was shown to reduce proliferation, whereas its overexpression increases proliferation. Together with the finding that growth factors regulate Sec16A levels and its dynamics on ERES, it is proposed that this protein acts as an integrator linking growth factor signaling and secretion. This provides a mechanistic basis for the previously proposed link between secretion and proliferation (Tillmann, 2015).

Sec16 influences transitional ER sites by regulating rather than organizing COPII

During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. These ideas were tested using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, the functional regions of Sec16 were dissected. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. It is proposed that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites (Bharucha, 2013).


REFERENCES

Search PubMed for articles about Drosophila Sec16

Aguilera-Gomez, A., van Oorschot, M. M., Veenendaal, T. and Rabouille, C. (2016). In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation. Elife 5. PubMed ID: 27874829

Aguilera-Gomez, A., Zacharogianni, M., van Oorschot, M. M., Genau, H., Grond, R., Veenendaal, T., Sinsimer, K. S., Gavis, E. R., Behrends, C. and Rabouille, C. (2017). Phospho-Rasputin stabilization by Sec16 is required for stress granule formation upon amino acid starvation. Cell Rep 20(4): 935-948. PubMed ID: 28746877 

Aulas, A., Stabile, S. and Vande Velde, C. (2012). Endogenous TDP-43, but not FUS, contributes to stress granule assembly via G3BP. Mol Neurodegener 7: 54. PubMed ID: 23092511

Bharucha, N., Liu, Y., Papanikou, E., McMahon, C., Esaki, M., Jeffrey, P. D., Hughson, F. M. and Glick, B. S. (2013). Sec16 influences transitional ER sites by regulating rather than organizing COPII. Mol Biol Cell 24(21): 3406-3419. PubMed ID: 24006484

Bhattacharyya, D. and Glick, B. S. (2007). Two mammalian Sec16 homologues have nonredundant functions in endoplasmic reticulum (ER) export and transitional ER organization. Mol Biol Cell 18(3): 839-849. PubMed ID: 17192411

Connerly, P. L., Esaki, M., Montegna, E. A., Strongin, D. E., Levi, S., Soderholm, J. and Glick, B. S. (2005). Sec16 is a determinant of transitional ER organization. Curr Biol 15(16): 1439-1447. PubMed ID: 16111939

Cook, M. S., Cazin, C., Amoyel, M., Yamamoto, S., Bach, E. and Nystul, T. (2017). Neutral competition for Drosophila follicle and cyst stem cell niches requires vesicle trafficking genes. Genetics [Epub ahead of print]. PubMed ID: 28512187

Di Paola, S., Micaroni, M., Di Tullio, G., Buccione, R. and Di Girolamo, M. (2012). PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ss1. PLoS One 7(6): e37352. PubMed ID: 22701565

Farny, N. G., Kedersha, N. L. and Silver, P. A. (2009). Metazoan stress granule assembly is mediated by P-eIF2alpha-dependent and -independent mechanisms. RNA 15(10): 1814-1821. PubMed ID: 19661161

Gardner, B. M., Pincus, D., Gotthardt, K., Gallagher, C. M. and Walter, P. (2013). Endoplasmic reticulum stress sensing in the unfolded protein response. Cold Spring Harb Perspect Biol 5(3): a013169. PubMed ID: 23388626

Gao, J., Schatton, D., Martinelli, P., Hansen, H., Pla-Martin, D., Barth, E., Becker, C., Altmueller, J., Frommolt, P., Sardiello, M. and Rugarli, E. I. (2014). CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins. J Cell Biol 207(2): 213-223. PubMed ID: 25349259

Goh, L. H., Zhou, X., Lee, M. C., Lin, S., Wang, H., Luo, Y. and Yang, X. (2013). Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains. Dev Biol 381(2): 353-364. PubMed ID: 23835532

Hughes H, Budnik A, Schmidt K, Palmer KJ, Mantell J, Noakes C, Johnson A, Carter DA, Verkade P, Watson P, Stephens DJ (2009) Organisation of human ER-exit sites: requirements for the localisation of Sec16 to transitional ER. J Cell Sci 122: 2924-2934. PubMed ID: 19638414

Ivan, V., de Voer, G., Xanthakis, D., Spoorendonk, K. M., Kondylis, V. and Rabouille, C. (2008). Drosophila Sec16 mediates the biogenesis of tER sites upstream of Sar1 through an arginine-rich motif. Mol Biol Cell 19(10): 4352-4365. PubMed ID: 18614796

Iwasaki, H., Yorimitsu, T. and Sato, K. (2017). Microscopy analysis of reconstituted COPII coat polymerization and Sec16 dynamics. J Cell Sci 130(17): 2893-2902. PubMed ID: 28747320

Jevtov, I., Zacharogianni, M., van Oorschot, M. M., van Zadelhoff, G., Aguilera-Gomez, A., Vuillez, I., Braakman, I., Hafen, E., Stocker, H. and Rabouille, C. (2015). TORC2 mediates the heat stress response in Drosophila by promoting the formation of stress granules. J Cell Sci 128(14): 2497-2508. PubMed ID: 26054799

Jwa, M. and Chang, P. (2012). PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK- and IRE1alpha-mediated unfolded protein response. Nat Cell Biol 14(11): 1223-1230. PubMed ID: 23103912

Kedersha, N., Panas, M. D., Achorn, C. A., Lyons, S., Tisdale, S., Hickman, T., Thomas, M., Lieberman, J., McInerney, G. M., Ivanov, P. and Anderson, P. (2016). G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits. J Cell Biol 212(7): 845-860. PubMed ID: 27022092

Patel, A., Lee, H. O., Jawerth, L., Maharana, S., Jahnel, M., Hein, M. Y., Stoynov, S., Mahamid, J., Saha, S., Franzmann, T. M., Pozniakovski, A., Poser, I., Maghelli, N., Royer, L. A., Weigert, M., Myers, E. W., Grill, S., Drechsel, D., Hyman, A. A. and Alberti, S. (2015). A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162(5): 1066-1077. PubMed ID: 26317470

Maeda, M., Katada, T. and Saito, K. (2017). TANGO1 recruits Sec16 to coordinately organize ER exit sites for efficient secretion. J Cell Biol 216(6): 1731-1743. PubMed ID: 28442536

Molliex, A., Temirov, J., Lee, J., Coughlin, M., Kanagaraj, A. P., Kim, H. J., Mittag, T. and Taylor, J. P. (2015). Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization. Cell 163(1): 123-133. PubMed ID: 26406374

Narayanaswamy, R., Levy, M., Tsechansky, M., Stovall, G. M., O'Connell, J. D., Mirrielees, J., Ellington, A. D. and Marcotte, E. M. (2009). Widespread reorganization of metabolic enzymes into reversible assemblies upon nutrient starvation. Proc Natl Acad Sci U S A 106(25): 10147-10152. PubMed ID: 19502427

Piao, H., Kim, J., Noh, S. H., Kweon, H. S., Kim, J. Y. and Lee, M. G. (2017). Sec16A is critical for both conventional and unconventional secretion of CFTR. Sci Rep 7: 39887. PubMed ID: 28067262

Sprangers, J. and Rabouille, C. (2015). SEC16 in COPII coat dynamics at ER exit sites. Biochem Soc Trans 43(1): 97-103. PubMed ID: 25619252

Tang, B. L. (2017). Sec16 in conventional and unconventional exocytosis: Working at the interface of membrane traffic and secretory autophagy? J Cell Physiol 232(12): 3234-3243. PubMed ID: 28160489

Tillmann, K. D., Reiterer, V., Baschieri, F., Hoffmann, J., Millarte, V., Hauser, M. A., Mazza, A., Atias, N., Legler, D. F., Sharan, R., Weiss, M. and Farhan, H. (2015). Regulation of Sec16 levels and dynamics links proliferation and secretion. J Cell Sci 128(4): 670-682. PubMed ID: 25526736

Tourriere, H., Chebli, K., Zekri, L., Courselaud, B., Blanchard, J. M., Bertrand, E. and Tazi, J. (2003). The RasGAP-associated endoribonuclease G3BP assembles stress granules. J Cell Biol 160(6): 823-831. PubMed ID: 12642610

Wang, Z. H., Rabouille, C. and Geisbrecht, E. R. (2015). Loss of a Clueless-dGRASP complex results in ER stress and blocks integrin exit from the perinuclear endoplasmic reticulum in Drosophila larval muscle. Biol Open. PubMed ID: 25862246

Wilhelmi, I., Kanski, R., Neumann, A., Herdt, O., Hoff, F., Jacob, R., Preussner, M. and Heyd, F. (2016). Sec16 alternative splicing dynamically controls COPII transport efficiency. Nat Commun 7: 12347. PubMed ID: 27492621

Zacharogianni, M., Kondylis, V., Tang, Y., Farhan, H., Xanthakis, D., Fuchs, F., Boutros, M. and Rabouille, C. (2011). ERK7 is a negative regulator of protein secretion in response to amino-acid starvation by modulating Sec16 membrane association. EMBO J 30(18): 3684-3700. PubMed ID: 21847093

Zacharogianni, M., Aguilera-Gomez, A., Veenendaal, T., Smout, J. and Rabouille, C. (2014). A stress assembly that confers cell viability by preserving ERES components during amino-acid starvation. Elife 3. PubMed ID: 25386913


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date revised: 12 October 2017

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