InteractiveFly: GeneBrief

Sac1 phosphatase: Biological Overview | References

Gene name - Sac1 phosphatase

Synonyms -

Cytological map position - 61F4-61F4

Function - enzyme

Keywords - PI4P phosphatase, lipid phosphatase, promotes microtubule stability in the developing retina, regulates postembryonic synaptic maturation and neurodegeneration, binding partner of VAMP-associated protein 33kDa (Vap33 or DVap), Drosophila model of amyotrophic lateral sclerosis, functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation, dorsal closure, axonal transport, microtubule stability, regulates vesicular trafficking

Symbol - Sac1

FlyBase ID: FBgn0283500

Genetic map position - chr3L:1,248,020-1,250,451

NCBI classification - Phosphoinositide polyphosphatase (Sac family)

Cellular location - cytoplasmic

NCBI link: EntrezGene, Nucleotide, Protein
Sac1 orthologs: Biolitmine

Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). This study using a novel temperature-sensitive (ts) allele, showed that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo. Together, these data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner (Del Bel, 2018).

Undifferentiated epithelial cells are patterned and specified during development to yield highly ordered tissues composed of multiple cell types. Patterning and differentiation within an epithelium are driven in part by cell surface adhesion molecules that promote intercellular interactions. Defects in cell-cell adhesion lead to developmental abnormalities and contribute to disease progression. For example, the mammalian Irre cell recognition module (IRM) adhesion proteins NEPH1 (KIRREL) and nephrin (NPHS1) are required in the kidney for development and maintenance of the filtration barrier or slit diaphragm. Despite their importance in animal development and physiology, little is known about how trafficking and delivery of cell surface adhesion molecules such as IRM proteins is achieved (Del Bel, 2018).

The adult Drosophila eye contains ~750 individual units called ommatidia. Prior to pupariation, each ommatidium consists of eight photoreceptor (PR) cells and four cone cells, surrounded by undifferentiated interommatidial cells (IOCs). During the first half of pupal eye development, 0-42 h after puparium formation (APF), IOCs undergo dynamic morphogenetic changes to give rise to two primary pigment cells (1°pc), six secondary pigment cells (2°pc), three tertiary pigment cells (3°pc) and three bristles, arranged in a honeycomb lattice. These cells support and optically isolate individual ommatidia (Del Bel, 2018).

Specification and organization of Drosophila retinal cells requires IRM protein function. 1°pc express the nephrin homologs Hibris (Hbs) and Sticks and stones (Sns), whereas IOCs express the NEPH1 homologs Roughest (Rst) and Kin of Irre (Kirre, also called Dumbfounded). Between 24 and 30 h APF, all four proteins localize to the plasma membrane (PM) of these cells at the 1°pc:IOC border, and heterophilic binding of Rst and Kirre with Hbs and Sns is needed for specification and morphogenesis of 2°pc and 3°pc (2°/3°pc). Mutations that affect cell surface accumulation of Rst result in rough eyes and reduced levels of pigmentation as a result of defects in 2°/3°pc differentiation (Del Bel, 2018).

Phosphoinositides, or phosphatidylinositol (PI) phosphates (PIPs), regulate essential cellular processes such as membrane trafficking and actin cytoskeletal organization. In the canonical PIP pathway, PI is phosphorylated by PI 4-kinases (PI4Ks) to generate PI 4-phosphate (PI4P), which is a precursor for other PIPs, including PI 4,5-bisphosphate [PI(4,5)P2], and serves as a potent signaling molecule, for example by recruiting effectors to the Golgi body to promote membrane trafficking. Downregulation of PI4P in specific membrane compartments also drives cellular events. For example, in budding yeast (Saccharomyces cerevisiae), PI4P dephosphorylation is required for targeted delivery of cargo to the PM by the exocyst complex. Drosophila has three PI4Ks, including a single type II enzyme (PI4KII) that generates PI4P at the trans-Golgi network (TGN) and on endosomes. PI4P levels are kept in check by the phosphoinositide phosphatase Sac1, a conserved transmembrane protein present in endoplasmic reticulum and Golgi membranes (Del Bel, 2018).

Previous work has shown that Sac1 is required for normal eye development in Drosophila (Wei, 2003b). Sac1 is essential for viability in Drosophila (Wei, 2003a,b). Flies transheterozygous for a hypomorphic allele and a null allele exhibit rough eyes with necrotic patches and drown in the food soon after eclosing (Wei, 2003b). Using the hypomorphic allele, which is shown to be temperature sensitive (ts), it was demonstrated that Sac1 plays a crucial role in patterning the retinal epithelium. IOCs of Sac1ts flies cultured at 25°C exhibit a dramatic increase in PI4P levels and decreased levels of PM PI(4,5)P2. Although Rst is present at the cell surface of these IOCs, fixation on ice ex vivo results in re-distribution of Rst to enlarged structures containing the exocyst complex subunit Sec8, suggesting a microtubule defect. Indeed, Sac1ts IOCs contain sparse, disorganized microtubules (MTs) that disappear when fixed on ice. These results thus identify a novel link between Sac1, PIP levels and microtubule stability in the developing Drosophila eye. Because Sac1 is conserved, these findings suggest a possible role for MT regulation by Sac1 in human development and disease (Del Bel, 2018).

Conditional alleles are powerful tools for studying the functions of essential genes. Using a temperature-sensitive allele of Sac1, this study shows that proper phospholipid regulation by Sac1 is required for normal development of the Drosophila retinal epithelium (Del Bel, 2018).

The results indicate that retinal patterning is highly sensitive to elevated levels of PI4P. Loss of Sac1 leads to increased Golgi PI4P and IOC patterning defects that are rescued by expression of catalytically active Sac1. Deletion of PI4KII suppresses Sac1ts, whereas expression of a WT PI4KII transgene exacerbates the observed defects. Thus, Sac1 and PI4KII regulate a pool of PI4P that is required for retinal patterning. Loss of PI4KII itself results in mild patterning defects (i.e., occasional extra bristles and fused ommatidia), indicating that reducing PI4P levels also has consequences for retinal development. Although these data point to a clear role for PI4KII in signaling required for 2°/3°pc adhesion and patterning, the experiments do not rule out possible contributions of other PI4Ks to this process. Moreover, it is anticipated that, in addition to the Golgi, other pools of PI4P at the PM and elsewhere may be regulated by Sac1 (Del Bel, 2018).

Because PI4P is phosphorylated by PIP 5-kinases to produce PI(4,5)P2, Sac1ts mutants might be expected to show increased levels of PI(4,5)P2 as well as PI4P. Strikingly, however, this study found that Sac1ts mutants exhibit reduced levels of the PI(4,5)P2 marker PLCΔ-PH at the PM. Although this result is counterintuitive, similar observations have been reported in budding yeast, and the same same phenomenon in Drosophila is observed embryos homozygous for a lethal allele of Sac1. This observation is in contrast to other Drosophila tissues, where Sac1 loss was reported to have no effect on PI(4,5)P2 levels (Forrest, 2013; Yavari, 2010). It is possible that PIP 5-kinases lack access to the increased PI4P in Sac1 mutants, thereby preventing the expected increase in PI(4,5)P2, or perhaps increased PI4P triggers a feedback mechanism that activates PIP phosphatases or phospholipases, leading to PI(4,5)P2 breakdown. Thus, in addition to elevated PI4P levels, reduced levels of PI(4,5)P2 might contribute to the IOC patterning defects in Sac1ts. Indeed, in preliminary results a genetic interaction was observed between Sac1 and the PI4P 5-kinase skittles, which could stem from reduced PI(4,5)P2 or increased PI4P levels, or a combination of the two (Del Bel, 2018).

Drosophila Sac1ts mutants exhibit rough eyes, ommatidial mispatterning and abnormal 2°/3°pc morphology, phenotypes that resemble mutants for the IRM protein Rst. Maintenance of Rst at the 1°pc:IOC border is sensitive to ice fixation in Sac1ts mutant retinas. Because MTs are labile under cold conditions, this suggested a MT defect in Sac1ts. MTs are disorganized in Sac1ts mutant IOCs fixed at RT, and even more dramatically affected in mutant IOCs fixed on ice. Moreover, Sac1ts IOCs fixed on ice display enlarged Sec8-positive vesicles containing Rst, suggesting that Rst travels to the cell surface via an exocyst- and MT-associated pathway that is sensitive to cold fixation in Sac1ts (Del Bel, 2018).

The simplest model to explain these data is that Rst is constantly turned over at the PM and requires continuous replenishment from internal stores, as has been reported for mammalian IRM proteins. This turnover appears to occur in both WT and Sac1ts, as this study observed similar uptake of anti-Rst antibody in each. It remains possible that subtle defects in the activity or turnover of Rst (or other proteins) at the PM contribute to the observed defects in Sac1ts. Indeed, compromised expression of the differentiation marker BA12-lacZ was observed in Sac1ts IOCs. Defects in BA12-lacZ expression, as well as IOC number and specification, also occur in RstD mutants, which show a delay in Rst accumulation at the 1°pc:IOC border. Relevant to this point, it is noted that when quantification of Rst distribution after RT or cold fixation was independently validated at a later date, a slight but significant reduction was observed in Rst accumulation in Sac1ts following RT fixation. However, as this difference was not observed in earlier experiments, it is not possible to conclude that there is a reproducibly significant decrease in Rst accumulation in Sac1ts (Del Bel, 2018).

Sac1 was originally identified as a suppressor of yeast actin mutants. Thus, much of the literature has focused on how Sac1 loss affects the actin cytoskeleton. This observed that retinal cells in Drosophila Sac1ts mutants exhibit normal actin organization during early stages of pupal eye development (24-30 h APF), yet show defects in MT stability and organization. Interestingly, both increased levels of PI4P and reduced levels of PI(4,5)P2 have previously been associated with defects in MT organization. The molecular mechanisms involved remain unknown, and further investigation will be required to determine the link between Sac1 and MT stability. However, the current observations suggest that regulation of the MT cytoskeleton by Sac1 may be more important when moving from fungal to animal models (Del Bel, 2018).

In summary, this study has characterized a requirement for Sac1 in maintaining PI4P and PI(4,5)P2 levels and promoting normal development of the Drosophila retina. Additionally, it was demonstrated that Rst distribution in Sac1ts is sensitive to cold fixation, and that Sac1ts exhibits a microtubule defect at 24 h APF. The results indicate that Rst is delivered to the PM via exocyst- and microtubule-based trafficking (which is cold sensitive in Sac1ts) and is turned over at the PM, similar to its mammalian homologs. Further investigation will be required to unravel precisely how Sac1 regulates microtubules, and how this contributes to normal retinal development (Del Bel, 2018).

Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1

The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (Sac1ts), this study shows that Sac1 is required for structural integrity of the Drosophila retinal floor. The βps-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in Sac1(ts) retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within Sac1(ts) retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in Drosophila retinal cells. Together, these data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development (Griffiths, 2020).

Although they comprise a minor fraction of total cellular phospholipid content, phosphoinositides, also known as phosphatidylinositol phosphates (PIPs), act as essential coordinators of membrane function and identity (Balla, 2013). PIPs are derived from the precursor phosphatidylinositol, whose inositol head group can be phosphorylated at any of three positions to yield seven unique PIP species that recruit distinct sets of effector proteins. Through the localized activity of PIP kinases and phosphatases, these species are interconverted to maintain enrichment in different membranes and to regulate numerous PIP effector-driven processes (Balla, 2013; Griffiths, 2020).

Sac1 is a conserved phosphatase whose substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates multiple stages in secretory trafficking, participates in cellular signaling pathways, and acts as the precursor for PI(4,5)P2 at the plasma membrane (PM). PI4P is produced in the PM and Golgi, respectively, by two conserved type III PI 4-kinases (PI4Ks), PI4KIIIα, and PI4KIIIβ. In addition, a type II PI4K (PI4KIIα) produces PI4P in the trans-Golgi network (TGN) and on endosomes, where it is important for endosomal trafficking (Griffiths, 2020).

In contrast to the distribution of PI4Ks and PI4P, Sac1 localizes primarily to the ER, as well as the cis-Golgi under growth-limiting conditions. Although seemingly capable of acting in trans on PI4P in neighboring membranes in some scenarios, Sac1 appears to predominantly depend on delivery of PI4P to the ER via nonvesicular lipid transport at membrane contact sites (MCS). For instance, oxysterol-binding protein (OSBP), which localizes to ER-trans-Golgi MCS through interactions with the ER-resident vesicle-associated membrane protein-associated protein VAP as well as PI4P in the trans-Golgi, delivers PI4P from the trans-Golgi to the ER in exchange for sterols. Hydrolysis of incoming PI4P by Sac1 maintains a low concentration of PI4P in the ER that is necessary for sustained PI4P/sterol countertransport in vitro, although this relationship appears more nuanced in vivo. OSBP-related proteins (ORPs), which are encoded by 11 genes in humans and three in flies, function similarly to OSBP but differ in their localization and lipid-binding preferences. Despite its essential function, how Sac1 regulates different aspects of cellular homeostasis during animal development is not fully understood (Griffiths, 2020).

In Drosophila, null Sac1 mutants exhibit embryonic lethality due to defects in cell shape and ectopically activated JNK signaling that prevent dorsal closure. JNK signaling defects are also observed in Sac1 clones in larval imaginal discs. Moreover, Sac1 regulates Hedgehog signaling by inhibiting recruitment and activation of Smoothened at the PM in a PI4P-dependent manner (Yavari, 2010; Jiang, 2016). Sac1 is also required for axonal pathfinding in the embryonic central nervous system, as well as for axonal transport and synaptogenesis in larval neurons (Lee, 2011; Forrest, 2013; Griffiths, 2020).

In addition, loss of Sac1 causes severe tissue disorganization and degeneration during eye development. The Drosophila eye is composed of ~750 unit eyes called ommatidia. Presumptive ommatidia arise early in pupal development, where they initially comprise clusters of medial/basal photoreceptors and apical cone cells surrounded by a disordered pool of undifferentiated interommatidial cells (IOCs). During the first half of the ∼96-h pupal stage, two IOCs per ommatidium differentiate into primary pigment cells (1°pc), which encircle the cone cells. The remaining IOCs subsequently differentiate into a lattice of secondary and tertiary pc (2°/3°pc) and sensory bristles that separate neighboring ommatidia or are removed by apoptosis by 42 h after puparium formation (APF). Changes in IOC shape and position during this stage require the Irre cell recognition module (IRM) adhesion proteins Roughest (Rst) and Hibris (Hbs), as well as their paralogues Kirre and Sticks and stones (Sns). Rst/Kirre and Hbs/Sns are orthologues of mammalian Neph1 and nephrin, which are needed for formation of the renal slit diaphragm as well as during myoblast fusion. After IOC patterning, during late stages of pupal eye development (42-96 h APF), the retina elongates fivefold, and laminated corneal lenses with underlying gelatinous pseudocones are secreted, giving the eye its characteristic adult appearance (Griffiths, 2020).

Previously work has examined the role of Sac1 in the developing Drosophila eye using a hypomorphic Sac1 allele that is temperature sensitive (Sac1ts). Sac1ts flies develop morphologically normal eyes when reared at 18°C, but display a rough eye phenotype caused by defective IOC sorting when reared at or above 23.5°C. This study shows that Sac1ts eyes exhibit structural defects at the retinal floor and mislocalization of the βps-integrin Myospheroid (Mys), which is required for retinal floor adhesion. This defect is not due to a loss of cell polarity, as apical adherens junctions are unaffected. However, a novel secondary defect was identified in the distribution of Rst and Kirre, which are apical transmembrane proteins. At 42 h APF, Sac1ts 2°/3°pc contain an excess of intracellular Rst and Kirre due to impaired endo-lysosomal trafficking and degradation. Sac1ts 2°/3°pc also accumulate PI4P and F-actin on enlarged, basal endosomes and exhibit ER stress. Thus, this study has identified novel roles for Sac1 in regulating cellular homeostasis during tissue morphogenesis (Griffiths, 2020).

The Drosophila pupal eye represents a powerful system to examine protein trafficking and turnover. Patterning of retinal cells requires spatially and temporally regulated expression as well as correct subcellular distribution of cell surface proteins that mediate cell-cell contacts and determine tissue architecture. Dysregulation of these processes can produce structural defects, which frequently persist in the adult eye. This study has taken advantage of these circumstances to demonstrate the importance of Sac1 in basal delivery of the βps-integrin Mys, which is required for retinal floor integrity, as well as endo-lysosomal regulation and turnover of the apical patterning determinants Rst and Kirre. The results also highlight the importance of Drosophila Sac1 in ER homeostasis, as had been reported in yeast. This could be due to deregulation of PI4P, phosphatidylserine, and sterol levels, which would be expected to disrupt ER membrane charge and lipid order (Griffiths, 2020).

Given the similarities between mys mutants and Sac1ts, loss of Mys at the basal grommets in Sac1ts likely causes the retinal floor defects observed in the adult eye. In addition, this phenotype resembles the basal retinal degeneration observed in an ALS-associated vap mutant, suggesting the underlying cause could be similar. However, it is unclear why basal distribution of Mys is perturbed while apical polarity is not. In the Drosophila follicular epithelium, Rab10 activity has been shown to be important for the distribution of basement membrane proteins independent of overall apical-basal polarity, in a manner dependent on PI(4,5)P2 at the apical PM. Previously study observed a decrease in apical PI(4,5)P2 abundance in Sac1ts retinas at 24 h APF (Del Bel, 2018), which it is speculated could perturb basal trafficking. Alternatively, aberrant distribution of basal F-actin in Sac1ts could inhibit localization of Mys to the grommets. Why some transmembrane proteins are sensitive to reduced Sac1 activity while others are not remains an open question. It is also unclear whether Mys mislocalization is linked to endosome dysfunction in Sac1ts (Griffiths, 2020).

Whereas PI3P and PI(3,5)P2 are the canonical phosphoinositide regulators of endosomal progression (Wallroth, 2018), PI4P production has also emerged as an important factor in cargo delivery to lysosomes. In mammalian cells, PI4P is generated on late endosomes by type II PI4Ks (Baba, 2019). PI4KIIα is important for Golgi-to-lysosome trafficking of LIMP-2, as well as PM-to-lysosome trafficking of LAMP-1, and these proteins accumulate in enlarged endosomes when PI4KIIα levels are reduced. Furthermore, in macrophages, PI4KIIα-mediated PI4P enrichment on phagosomes occurs concurrently with Rab7 recruitment and is necessary for phagosome acidification and subsequent fusion with lysosomes (Griffiths, 2020).

This study has shown that Sac1-dependent depletion of PI4P is also important for endosomal trafficking and degradation of transmembrane proteins from the PM. This is consistent with a recent report by Mao and colleagues (Mao, 2019), who found that in multiple larval Drosophila tissues, loss of VAP, which recruits OSBP and a subset of ORPs to MCS, increases endosomal PI4P levels and inhibits autophagic degradation. Null vap mutants exhibit decreased lysosomal acidification, as well as an increase in the abundance of lysosomes, endosomes, autolysosomes, autophagosomes, and Ref(2)P (Mao, 2019). The authors propose that increased PI4P abundance up-regulates endosome formation and progression, which causes lysosomes to become oversaturated with incoming cargo. Indeed, loss of Ubiquilin, which contributes to lysosome acidification, also delays autophagy and causes Ref(2)P buildup. Notably, this study observed increased Ref(2)P abundance in Sac1ts retinas, which suggests a similar delay in autophagy. It is a compelling notion that increased PI4P levels in Sac1ts could promote excessive fusion of endosomes with lysosomes, which would replicate the effect described by Mao (2019). However, the accumulation of Rst and Kirre in Sac1ts, which do not appear to be concentrated in lysosomes based on the lack of colocalization between Rst and Arl8, could also be caused by impaired endosomal progression or maturation, though this might stem from downstream lysosomal dysfunction. Indeed, the enlarged endosomes observed in Sac1ts lacked both Vps16a and Arl8, suggesting they were not caused by excessive fusion with lysosomes. Further analysis of PI4P in endosomal dynamics and maturation is warranted to determine the precise role of Sac1 in late stages of protein degradation (Griffiths, 2020).

This study also found that reduced Sac1 function leads to basal accumulation of F-actin-positive enlarged endosomes. In mammalian cells, loss of both VAP isoforms has been shown to induce F-actin comet formation on endosomes via PI4P-dependent recruitment of the WASH-ARP2/3 complex. Notably, these do not resemble the more uniform F-actin coating on Sac1ts endosomes. Rather, the structures that were observed appear more reminiscent of a phenomenon termed actin-flashing, wherein phagosomes become coated in F-actin by WASP-ARP2/3 to delay fusion with lysosomes. Endosomal phenotypes similar to those in Sac1ts have also been observed when Arf6 activity is perturbed; increased Arf6 activity activates PIP5K, which has been shown to produce PI(4,5)P2 on endosomes and lead to F-actin polymerization via WASP, whereas loss of Arf6 increases endosomal PI4P levels and perturbs endosomal recycling. Intriguingly, in Caenorhabditis elegans, Sac1 inhibits Arf6 by sequestering the Arf6-GEF Bris-1. However, it is unknown whether this interaction is conserved or, more broadly, how Sac1 influences F-actin polymerization on endosomes (Griffiths, 2020).

It is noteworthy that enlarged endosomes were restricted to basal regions in Sac1ts. Positioning of endosomes and lysosomes is mediated by bidirectional transport along microtubules, which influences their acidity and function. In mammalian cells, Rab7 recruits RILP, which activates endosomal dynein motors to promote minus end-directed transport toward perinuclear microtubule organizing centers. PI4P is also required for RILP recruitment, which implies that excess PI4P could lead to perinuclear endosome accumulation. Although the single Drosophila RILP orthologue has been shown to bind Arl8 rather than Rab7, it is possible that PI4P influences late endosome transport through analogous Rab7 effectors. Additionally, previous work has shown that Sac1ts 2°/3°pc precursors contain unstable microtubules at 24 h APF (Del Bel, 2018), which could affect microtubule-based endosome positioning later in development (although this study was unable to detect microtubule defects by immunostaining at 42 h APF). However, it is also possible that enlarged endosomes accumulate basally for other reasons or are simply excluded from narrower apical-medial regions on the basis of size. It remains to be discerned whether Rst accumulation and the appearance of enlarged endosomes, which co-occurred between 24 and 42 h APF, share a causal basis or represent distinct, parallel phenotypes of reduced Sac1 activity (Griffiths, 2020).

Given the phenotypic similarities between Sac1ts and vap mutants (Mao, 2019), it was surprising that osbp did not affect Mys distribution or cause severe Rst accumulation. However, this is reminiscent of previous results from Drosophila neurons, where loss of Vap but not OSBP caused protein accumulation and ER stress (Moustaqim-Barrette, 2014). It is possible that, as in yeast where the presence of one out of seven OSBP homologues is sufficient for viability, OSBP functions redundantly with one or more ORPs in regulating the endosomal pathway. Indeed, CG1513, which is synthetically lethal in combination with osbp (Moustaqim-Barrette, 2014), encodes an orthologue of mammalian ORP9, which functions similarly to OSBP in sterol-PI4P exchange at ER-Golgi MCS. Alternatively, CG3860 encodes an orthologue of mammalian ORP2, which localizes to late endosomes in HeLa cells and influences sterol levels in endosomes and the PM, although countertransport of PI4P has not been shown. Mammalian ORP2 also binds ORP1L, which acts at ER-endosome MCS and promotes endosome transport, though it is unclear whether such a role is conserved in Drosophila, which lack an ORP1L orthologue. Further characterization of the Drosophila ORPs is thus needed to clarify their respective contributions to lipid homeostasis and endosomal progression (Griffiths, 2020).

Recent years have seen a proliferation of research into Sac1's roles in lipid homeostasis and the importance of PI4P regulation, as well as the development of novel probes and methods for studying phosphoinositides in vivo. This study has provided new insights into Sac1's function in protein delivery and turnover in a developing tissue, which will serve as groundwork for further investigations into the significance of Sac1 in cell physiology, organismal development, and ultimately cellular homeostasis in human health and disease (Griffiths, 2020).

Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis

The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. This study has identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, this study found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, this study reports that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP-Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS (Forrest, 2013).

Amyotrophic lateral sclerosis (ALS) is a progressive, degenerative disorder characterized by the selective loss of motor neurons in the brain and spinal cord leading to paralysis, muscle atrophy and eventually, death. Two missense mutations in the gene encoding the human Vesicle-associated membrane protein (VAMP)-Associated Protein B (hVAPB) causes a range of dominantly inherited motor neuron diseases including ALS8. VAP family proteins are characterized by an N-terminal major sperm protein (MSP) domain, a coiled-coil (CC) motif and a transmembrane (TM)-spanning region. They are implicated in several biological processes, including regulation of lipid transport, endoplasmic reticulum (ER) morphology and membrane trafficking. Drosophila Vap-33-1 (hereafter, DVAP) regulates synaptic structure, synaptic microtubule (MT) stability and the composition of postsynaptic glutamate receptors (Chai, 2008; Pennetta, 2002). MSP domains in DVAP are cleaved and secreted into the extracellular space where they bind Ephrin receptors. MSPs also bind postsynaptic Roundabout and Lar-like receptors to control muscle mitochondria morphology, localization and function. Transgenic expression of the disease-linked alleles (DVAP-P58S and DVAP-T48I) in the larval motor system recapitulates major hallmarks of the human disease, including aggregate formation, locomotion defects and chaperone upregulation. Several studies have also implicated the ALS mutant allele in abnormal unfolded protein response (UPR) and in the disruption of the anterograde axonal transport of mitochondria. However, it is unclear how these diverse VAP functions are achieved and which mechanisms underlie the disease pathogenesis in humans. One way to address these questions is to search for DVAP-interacting proteins. This study identified Sac1 (Suppressor of Actin 1), an evolutionarily conserved phosphoinositide phosphatase, as a DVAP-binding protein. Phosphoinositides are low-abundance lipids that localize to the membrane-cytoplasm interface and function by binding various effector proteins. The inositol group can be reversibly phosphorylated at the 3', 4' and 5' positions to generate seven possible phosphoinositide derivatives, each with a specific intracellular dynamic distribution. Sac1 predominantly dephosphorylates PtdIns4P pools, although PtdIns3P and PtdIns(3,5)P2 can also function as substrates. In yeast, Sac1 has been linked to several processes, including actin organization, vacuole morphology and sphingomyelin synthesis. Drosophila Sac1 mutants die as embryos and exhibit defects in dorsal closure and axonal pathfinding. Mouse lines deficient for Sac1 are cell lethal, whereas Sac1 downregulation in mammalian cell cultures results in disorganization of Golgi membranes and mitotic spindles. Interestingly, SAC3 (also known as FIG4), another member of the Sac phosphatase family, is mutated in familial and sporadic cases of ALS. Inactivation of SAC3 in mice also results in extensive degeneration and neuronal vacuolization in the brain, most relevantly in the motor cortex. This study identified Sac1 and DVAP as binding partners and shows that DVAP is required to maintain normal levels of PtdIns4P. Loss of either Sac1 or DVAP function disrupts axonal transport, MT stability, synaptic growth and the localization of a number of postsynaptic markers. The disease-causing mutation (DVAP-P58S) induces neurodegeneration and displays synaptic phenotypes similar to those of either Sac1 or DVAP loss-of-function, including an increase in PtdIns4P levels. Importantly, reducing PtdIns4P levels rescues the neurodegeneration associated with DVAP-P58S and suppresses the synaptic phenotypes associated with DVAP-P58S and DVAP loss-of-function alleles. Consistent with these observations, Sac1 is sequestered into DVAP-P58S-mediated aggregates and downregulation of Sac1 in neurons induces increased PtdIns4P levels and degeneration. These data highlight the crucial role of DVAP and Sac1 in regulating phosphoinositides and support a causative role for PtdIns4P levels in ALS8 pathogenesis (Forrest, 2013).

This study has identified Sac1 as a DVAP-binding protein and uncovered a hitherto unknown function of Sac1 in postembryonic synaptic maturation and neurodegeneration. Presynaptic reduction of either DVAP or Sac1 levels induces structural changes, disruption of the synaptic MT cytoskeleton and accumulation of clusters of proteins and vesicles along the axons. In addition, muscle down-regulation of either Sac1 or DVAP leads to a strikingly aberrant synaptic morphology and abnormal localization and distribution of several postsynaptic markers, including adducin and β-spectrin. Depletion of DVAP as well as Sac1 expression induces an increase in PtdIns4P levels. Sac1 downregulation in the adult nervous system was shown to cause early death and neurodegeneration in a dosage-dependent manner, a phenotype similar to that of DVAP-P58S transgenic expression. This analysis indicates that the DVAP-P58S allele has a dominant negative effect, as its transgenic expression leads to an upregulation of PtdIns4P and its mutant phenotypes are similar to those associated with either DVAP or Sac1 loss-of-function. In agreement with the hypothesis that neurodegeneration in the DVAP-P58S context is due to a loss-of-function of both DVAP and Sac1, it is reported that both wild-type DVAP and Sac1 are depleted from their normal localization and are sequestered into DVAP-P58S-mediated aggregates. Altogether, these data are consistent with a model in which DVAP is required for Sac1 activity and for the regulation of intracellular PtdIns4P levels. Loss-of-function of DVAP and Sac1 by a DVAP-P58S-mediated dominant-negative mechanism induces cell degeneration by an upregulation of PtdIns4P, which is also responsible for the observed disruption of fundamental biological processes at the NMJs . It has been previously shown that transgenic expression of DVAP proteins carrying the equivalent ALS8 mutations in Drosophila mimic the human disease. Notably, expression of hVAPB in flies rescues the lethality and the phenotypes associated with DVAP mutants, indicating an evolutionarily conserved function for VAP proteins. Collectively, these data indicate that DVAP-mediated molecular pathways are likely to be important for understanding of the disease pathogenesis in humans (Forrest, 2013).

There is evidence supporting that DVAP functions to maintain normal cellular levels of PtdIns4P by interacting with Sac1. First, Sac1 and DVAP bind to each other and colocalize in many different tissues. It has been reported that phosphoinositol transfer proteins/phosphoinositide-binding proteins associate directly with phosphatases and kinases to control their activities. Specifically, VAP has been shown to bind PtdIns4P in vitro and to be required for Sac1 activity in yeast. Second, PtdIns4P levels are upregulated in DVAPRNAi mutants, suggesting that DVAP function is required for normal PtdIns4P levels. Similarly, in yeast, inactivation of Scs2/Scs22 VAP genes induces an increase in the levels of PtdIns4P. Third, the phenotypic similarity associated with either DVAP or Sac1 loss-of-function mutations supports the idea that the pool of PtdIns4P that is upregulated in DVAP mutants is the same as the one dephosphorylated by Sac1. Previous studies attributed a prominent functional role to the N-terminal MSP domain of DVAP. The MSP domain is cleaved and secreted and binds to the extracellular domain of Ephrin receptors. Secreted MSP also binds to Robo and Lar-like receptors to control mitochondria morphology, localization and function in muscles. A new DVAP-binding activity was identified that is MSP-independent and involves a C-terminal fragment encompassing the TM domain. Interestingly, a new hVAPB mutation replacing valine at position 234 with an isoleucine in the conserved TM domain of hVAPB has been shown to cause ALS8 in humans. These data may provide direct evidence of a role of hVAPB-Sac1 interaction in the disease pathogenesis in humans (Forrest, 2013).

In yeast and mammalian cells, Sac1 is an integral membrane protein localized to the ER and the Golgi. This study reports a similar localization for the Drosophila homologue of Sac1. In yeast and mammalian cells, Sac1 localization appears to be very dynamic, as this protein shuttles between ER and Golgi upon nutrient conditions. Specifically, glucose starvation in yeast or growth factor deprivation in mammalian cells causes relocalization of Sac1 from the ER to the Golgi complex, where it reduces PtdIns4P levels and slows protein trafficking. The ER-Golgi shuttling ability of Sac1 is reversed when nutrients or growth factors are added back to the growth medium. The growth factor-induced translocation of Sac1 from the Golgi to the ER requires p38 MAPK (mitogen-activated protein kinase) activity. These data suggest that Sac1 trafficking may be regulated by stressors that activate p38 MAPK. Some of these stressors such as oxidative damage and ER stress are triggers of neurodegeneration. This raises the intriguing possibility that a p38 MAPK-activated mechanism of PtdIns4P spatial regulation may be implicated in neurodegenerative processes (Forrest, 2013).

Scs2/Scs22 VAP proteins in yeast play a pivotal role in tethering the ER to the PM to form ER/PM contact sites. Studies have highlighted the role of membrane junctions between organelles as important sites for lipid metabolism and intracellular signalling controlled by PtdIns4P. Depletion of Scs2/Scs22 VAP proteins located to the ER/PM contact sites leads to a retraction of the ER into internal structures, elevated levels of PtdIns4Ps at the PM and induction of the UPR. At the ER/PM contact sites, Sac1 dephosphorylates PtdIns4P on the PM in trans from the ER. This reaction requires the Scs2/Sc22p VAP proteins and the oxysterol-binding homology proteins that act as PtdIns4P sensors and activates Sac1 phosphatase activity. ER/PM junctions have been described in many organisms and cell types, including neurons and Drosophila photoreceptors. In addition, VAP proteins have been implicated in ER-Golgi, ER-endosomes and ER-mitochondria contacts in mammalian cells, suggesting that they may function as a tether for several organelle/membrane contact sites. In conclusion, emerging evidence suggests that VAP proteins may be a crucial component of a hub controlling PtdIns4P metabolism in yeast and possibly, in higher eukaryotes as well (Forrest, 2013).

The ability of either PI4KIIIα or four wheel drive fwd, a Golgi-localized lipid kinase that synthesizes phosphatidylinositol 4-phosphate from phosphatidylinositol, to suppress the synaptic and neurodegenerative phenotypes associated with transgenic expression of DVAP-P58S is somewhat surprising, as their yeast homologues (Stt4 and Pik1, respectively) are supposed to play non-redundant functions and to control spatially separate pools of PtdIns4P. This is based on previously published data showing that, in yeast, Stt4 and Pik1 are both essential for cell viability but control different cellular processes. Pik1 is essential for anterograde vesicular trafficking, whereas Stt4 plays a role in actin cytoskeleton organization and protein kinase C signalling. Both Pik1 and Stt4 play distinct roles in regulating MAPK signalling. Localization studies further suggest that Pik1p is primarily present in the nucleus and in the Golgi, whereas Stt4p is mainly cytoplasmic and is recruited to the PM for localized synthesis of PtdIns4P (Forrest, 2013).

However, at present, the precise degree to which Stt4 and Pik1 functions have been conserved and apportioned among their homologues in flies remains unclear. In Drosophila, the Stt4 homologue PI4KIIIα is required for oocyte polarization and its intracellular localization has not been determined. On the other hand, previous studies revealed that the fly Pik1 homologue Fwd is required for male germ-line cytokinesis. In spermatocytes, Fwd localizes to the Golgi and it is required for the accumulation of PtdIns4P on this organelle, implying that its function in providing PtdIns4P in the Golgi is evolutionarily conserved with yeast. However, whereas Pik1 is required for cell viability, Fwd appears to be dispensable for normal development, suggesting that it is redundant with similar genes in carrying out its function (Forrest, 2013).

Another way to explain the rescue data would be to admit that upregulation of PtdIns(4,5P)2 and not PtdIns4P is responsible for DVAP-P58S mutant phenotypesPtdIns4P formed by the PM-associated STT4 can function as a substrate of PI4P 5-kinase to generate PtdIns(4,5)P2 at the cell cortex. It is also possible that PtdIns4P that is phosphorylated by plasmalemmal PI4P 5-kinase originates from intracellular sources. In the Golgi, PtdIns4P levels play a central role in the formation of vesicles delivered from the trans-Golgi network to the PM and their lipid cargo could be the substrate for the plasmalemmal PtdIns(4,5)P2 synthesis (676767). As PtdIns4P in the Golgi is mainly produced by Pik1 is therefore possible that PtdIns(4,5)P2 associated with the PM and its effector proteins are downstream of both Stt4 and Pik1. Moreover, upregulation of PtdIns(4,5)P2 would explain the MT phenotypes, the mislocalization of post-synaptic markers and the axonal transport defects. Indeed, PtdIns(4,5)P2-enriched microdomains in the PM have been shown to participate in the regulation of MT plus-end capture and stabilization during polarized mobility. In Caenorhabditis elegans, the microtubular motor UNC-4 gene was shown to be anchored to synaptic vesicles, using a pleckstrin homology domain, thus implicating PtdIns(4,5)P2 in MT-based intracellular motility . Finally, spectrin proteins and adducin require PtdIns(4,5)P2 for their correct localization to the cell cortex (Forrest, 2013).

However, if this were true, an increase in PtdIns(4,5)P2 levels whould be observed wherever an upregulation of PtdIns4P is observed. By using an antibody specific for PtdIns(4,5)P2, PtdIns(4,5)P2 levels were quantified in tissues in which either Sac1 or DVAP were downregulated as well as in tissues expressing the DVAP-P58S transgene. Surprisingly, PtdIns(4,5)P2 levels were not affected by the dramatic upregulation of PtdIns4P in any of the genotypes described earlier. Consistent with these data, it was previously reported that, in Drosophila eye imaginal discs, depletion of Sac1 exhibits a dramatic increase in PtdIns4P levels, whereas PtdIns(4,5)P2 and PtdIns3P levels remain similar to wild-type. In addition, loss of PM PtdIns4P by downregulation of PI4KIIIα was not matched by a decrease in PtdIns(4,5)P2 levels. It was shown that the major function of PtdIns4P is not to generate the pool of PtdIns(4,5)P2 on the PM but rather to contribute to the generation of a polyanionic lipid environment in the inner leaflet of the PM. PtdIns4P would then function in recruiting soluble proteins to the PM by electrostatic interaction with their polycationic surface. It was also shown that PtdIns4P contributes to processes such as modulation of ion channel activity that have been traditionally associated with changes in PtdIns(4,5)P2. Finally, upregulation of PtdIns(4,5)P2 by inactivation of the Drosophila PI(4,5)P2 5-phosphatase synaptojanin leads to a distinct endocytotic phenotype due to defects in synaptic vesicle recycling. In synaptojanin mutants, synaptic vesicles are severely depleted and those remaining are clearly clathrin-coated. Intracellular recordings revealed enhanced synaptic depression during prolonged high-frequency stimulation. Ultrastructural and electrophysiological analysis of DVAP mutants do not exhibit a synaptojanin-like phenotype, indicating that upregulation of PtdIns(4,5)P2 does not mimic the phenotype associated with increased levels of PtdIns4P. Taken together, these considerations suggest that increased levels of PtdIns4P could be the main factor determining the observed synaptic and neurodegenerative phenotypes. Further studies using fluorescent phosphoinositide probes and genetic analyses will be needed to fully clarify the contribution of PtdIns4P versus PtdIns(4,5)P2 pools to NMJ physiology and neurodegeneration (Forrest, 2013).

Emerging evidence indicates that VAP and Sac1 may also play an important and specific role in membrane homeostasis. Biogenesis of sphingolipids, sterols and phosphoinositides that together determine the structural and functional properties of cell membranes must be closely coordinated. VAP interacts with both oxysterol-binding protein (OSBP) and ceramide transfer protein (CERT), recruiting them to contact sites between the ER and the Golgi complex. CERT has a FFAT (diphenylalanine in an acidic tract) motif that mediates its binding to ER-localized VAP and a PH domain that recognizes the PtdIns4P-enriched Golgi membrane. It has been proposed that CERT, because of its dual-binding ability, shuttles ceramide from the ER to the Golgi, where it is converted into sphingomyelin. Sphingomyelin continues to move through the secretory pathway to the PM, where it is most abundant. OSBP has an analogous function to CERT but instead mediates inter-membrane sterol transfer. This functional similarity is also reflected in OSBP's domain architecture: like CERT, it contains a PtdIns4P-binding PH domain and a VAP-binding FFAT motif. It has been shown that sterols regulate sphingolipid metabolism by inducing a significant increase in SM synthesis that is dependent on OSBP, CERT and their shared binding partner VAP. The precise mechanism is not yet known but OSBP appears to activate CERT by promoting its recruitment to membranes and its binding to VAP. It is likely that disruption of the VAP-Sac1 interaction may have profound effects on the lipid composition of the PM, affecting its curvature and thickness and by consequence, vesicle budding and membrane remodelling. Interestingly, synaptic growth requires membrane remodelling and, at the Drosophila NMJs, occurs mainly by the budding of new boutons from pre-existing ones (Forrest, 2013).

VAPB is involved in the IRE1/XBP1 signalling pathway of the UPR, an ER reaction inhibiting the accumulation of unfolded/misfolded proteins. In the disease-context, the hVAPB-P56S protein recruits its wild-type counterpart into the aggregates and it attenuates its ability to induce the UPR. This together with the observation that, in yeast, depletion of VAP proteins from the ER/PM contact sites induces a constitutive activation of the UPR suggests that motor neurons in ALS8 could be particularly vulnerable to cell death-induced ER stress. In addition, VAP proteins have been shown to be involved in lipid transfer and metabolism and accumulation of lipids and intermediates of lipid biosynthetic pathways are potent inducers of apoptosis. Finally, recent studies in yeast have shown that defects in the PtdIns4K Pik1 activity lead to a blockage of autophagy, a process controlling the degradation of long-lived proteins, damaged organelles and bulk cytoplasm in response to various types of stress. Many questions remain to be explored concerning the precise molecular mechanism underlying neurodegeneration in ALS. However, over the last few years, an increasing number of experimental models have been generated and they represent an excellent tool for identifying molecular pathways in ALS and for evaluating their contribution to the disease pathogenesis (Forrest, 2013).

The phosphoinositide phosphatase Sac1 is required for midline axon guidance

Sac1 phosphoinositide (PI) phosphatases are important regulators of PtdIns(4)P turnover at the ER, Golgi, and plasma membrane (PM) and are involved in diverse cellular processes including cytoskeletal organization and vesicular trafficking. This study presents evidence that Sac1 regulates axon guidance in the embryonic CNS of Drosophila. Sac1 is expressed on three longitudinal axon tracts that are defined by the cell adhesion molecule Fasciclin II (Fas II). Mutations in the sac1 gene cause ectopic midline crossing of Fas II-positive axon tracts. This phenotype is rescued by neuronal expression of wild-type Sac1 but not by a catalytically-inactive mutant. Finally, sac1 displays dosage-sensitive genetic interactions with mutations in the genes that encode the midline repellent Slit and its axonal receptor Robo. Taken together, these results suggest that Sac1-mediated regulation of PIs is critical for Slit/Robo-dependent axon repulsion at the CNS midline (Lee, 2011).

Role of lipid metabolism in smoothened derepression in hedgehog signaling

The binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. This study shows that Smo activation is dependent on the levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). Loss of STT4 kinase, which is required for the generation of PI4P, exhibits hh loss-of-function phenotypes, whereas loss of Sac1 phosphatase, which is required for the degradation of PI4P, results in hh gain-of-function phenotypes in multiple settings during Drosophila development. Furthermore, loss of Ptc function, which results in the activation of Hh pathway, also causes an increase in PI4P levels. Sac1 functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation. Taken together, these results suggest a model in which Ptc directly or indirectly functions to suppress the accumulation of PI4P. Binding of Hh to Ptc derepresses the levels of PI4P, which, in turn, promotes Smo activation (Yavari, 2010).

A major regulatory step in the modulation of Hedgehog signaling occurs at the level of the two multipass transmembrane proteins, Patched and Smoothened. Genetic and biochemical studies suggest that the ligand Hh binds Ptc and functions in its inactivation. This inhibitory step is critical for the activation of Smo, which transduces the signal intracelluarly to promote Hh target gene activation. The importance of this regulatory step is further underscored by the observation that the Ptc/Smo interaction is the most commonly disrupted step in cancers caused upon aberrant Hh signaling (Yavari, 2010).

This article shows that phospholipid metabolism plays an important role in the modulation of Hh signaling at the level of Ptc/Smo interaction. In particular, the results show that an increase in the level of PI4P by the inactivation of Sac1 phosphatase leads to Smo protein relocalization to the membrane and an increase in Hh signaling in multiple tissues during Drosophila development. Furthermore the kinase (STT4), which is required for the generation of PI4P, is also required for the proper transduction of Hh signaling as indicated by its effects on Hh target gene expression. PI4P accumulation in the cell is a hallmark of sac1 mutations and is also seen upon loss of ptc activity. Furthermore, in sac1 mutant tissue, both increased membrane localization of Smo and accumulation of PI4P were found, whereas reduction in the PI4P kinase function leads to an hh-like loss of function phenotype. These results establish that phospholipid metabolism provides a critical regulatory input in the modulation of Hh signaling (Yavari, 2010).

Recent studies have proposed that Smo activation requires an input from a nonprotein small molecule. Cholesterol and its derivatives (oxysterols) are likely candidates for the small molecules required directly or indirectly for Ptc inhibition or Smo activation, because they also promote the translocation of Smo to the cilium. Because oxysterols are known to bind to vesicular transport proteins that also interact with phospholipids, further studies on possible cooperation between these two lipid types could further shed light the mechanism of Smo activation (Yavari, 2010).

Inactivation of Smo by Ptc occurs in a catalytic fashion in that a small number of Ptc molecules can inactivate many more Smo molecules. The current results provide an explanation for this nonstoichiometric inhibitory mechanism. The finding that inactivation of Ptc increases PI4P suggests that Ptc normally functions in keeping PI4P levels low within a cell. This could be achieved either by the down-regulation of the STT4 kinase or by the up-regulation of the Sac1 phosphatase. It is less likely that Ptc modulates Sac1 activity because in vivo localization studies in multiple models system have shown that Sac1 is predominantly localized to the Golgi and, as a result of proximity arguments alone, it seems a more likely possibility that Ptc modulates PI4P levels by down-regulating the lipid kinase. In this model, during normal Hh signaling, binding of Hh to Ptc will relieve repression of the kinase by Ptc and cause an increase in PI4P. As with all genetic analysis in Drosophila, the results do not imply direct protein interactions; currently unknown transduction components could exist, and future biochemical analyses will reveal which, if any, of the interactions is direct. However, the genetic analysis does allow a proposal of how an increase in the levels of this lipid can activate Hh signaling. Studies from both flies and vertebrate model system have suggested that the localization of Smo protein to the plasma membrane is essential for the activation of the pathway, and studies in multiple model systems have shown that PI4P function is essential in the vesicular transport of cargo proteins from the Golgi to the plasma membrane. It is therefore proposed that Hh binding to Ptc releases inhibition of a lipid kinase such as STT4, resulting in high PI4P levels. This aids vesicular transport of Smo to the membrane and causes its activation. A schematic representing the genetic model that is consistent with past and present data is shown in the graphical abstract. The results using Shh-responsive mouse fibroblasts indicate that mammalian Hh signal transduction is dependent on the activity of the murine STT4 ortholog. Previous localization studies suggest the STT4 ortholog contributes to plasma membrane PI4P pools, an observation consistent with a conserved role for PI4P metabolites in the control of Smo by mammalian Ptc1. The observation that RNAi against the mammalian PIK1 homolog, PI4III kinase α, also reduces Hh signal transduction could suggest it has diverged in function between flies and mammals. Alternatively, PI4P pools could be exchanged more readily between membrane-bound subcellular compartments and the cell surface in mammalian cells, making the removal of either of the PI4III kinases affect global availability of PI4P derivatives. In mammalian cells, Smo activation is associated with translocation of the molecule to the primary cilium, a ubiquitous microtubule-based cell surface protrusion. Given that Drosophila cells appear to lack primary cilia, it will be of interest to determine whether PI4III kinase activity is required for Smo translocation (Yavari, 2010).

The Sac1 lipid phosphatase regulates cell shape change and the JNK cascade during dorsal closure in Drosophila

The Sac1 lipid phosphatase dephosphorylates several phosphatidylinositol (PtdIns) phosphates and, in yeast, regulates a diverse range of cellular processes including organization of the actin cytoskeleton and secretion. This study has identified mutations in the gene encoding Drosophila Sac1. sac1 mutants die as embryos with defects in dorsal closure (DC). DC involves the migration of the epidermis to close a hole in the dorsal surface of the embryo occupied by the amnioserosa. It requires cell shape change in both the epidermis and amnioserosa and activation of a Jun N-terminal kinase (JNK) MAPK cascade in the leading edge cells of the epidermis. Loss of Sac1 leads to the improper activation of two key events in DC: cell shape change in the amnioserosa and JNK signaling. sac1 interacts genetically with other participants in these two events, and the data suggest that loss of Sac1 leads to upregulation of one or more signals controlling DC. This study is the first report of a role for Sac1 in the development of a multicellular organism.

Functions of Sac1 orthologs in other species

The activity of Sac1 across ER-TGN contact sites requires the four-phosphate-adaptor-protein-1

Phosphatidylinositol-4-phosphate (PI4P), a phosphoinositide with key roles in the Golgi complex, is made by Golgi-associated phosphatidylinositol-4 kinases and consumed by the 4-phosphatase Sac1 that, instead, is an ER membrane protein. This study shows that the contact sites between the ER and the TGN (ERTGoCS) provide a spatial setting suitable for Sac1 to dephosphorylate PI4P at the TGN. The ERTGoCS, though necessary, are not sufficient for the phosphatase activity of Sac1 on TGN PI4P, since this needs the phosphatidyl-four-phosphate-adaptor-protein-1 (FAPP1). FAPP1 localizes at ERTGoCS, interacts with Sac1, and promotes its in-trans phosphatase activity in vitro. It is envisioned that FAPP1, acting as a PI4P detector and adaptor, positions Sac1 close to TGN domains with elevated PI4P concentrations allowing PI4P consumption. Indeed, FAPP1 depletion induces an increase in TGN PI4P that leads to increased secretion of selected cargoes (e.g., ApoB100), indicating that FAPP1, by controlling PI4P levels, acts as a gatekeeper of Golgi exit (Venditti, 2019).


Search PubMed for articles about Drosophila Sac1 phosphatase

Balla, T. (2013). Phosphoinositides: tiny lipids with giant impact on cell regulation. Physiol Rev 93(3): 1019-1137. PubMed ID: 23899561

Baba, T., Toth, D. J., Sengupta, N., Kim, Y. J. and Balla, T. (2019). Phosphatidylinositol 4,5-bisphosphate controls Rab7 and PLEKHM1 membrane cycling during autophagosome-lysosome fusion. EMBO J 38(8): e100312. PubMed ID: 31368593

Chai, A., Withers, J., Koh, Y. H., Parry, K., Bao, H., Zhang, B., Budnik, V. and Pennetta, G. (2008). hVAPB, the causative gene of a heterogeneous group of motor neuron diseases in humans, is functionally interchangeable with its Drosophila homologue DVAP-33A at the neuromuscular junction. Hum Mol Genet 17(2): 266-280. PubMed ID: 17947296

Del Bel, L. M., Griffiths, N., Wilk, R., Wei, H. C., Blagoveshchenskaya, A., Burgess, J., Polevoy, G., Price, J. V., Mayinger, P. and Brill, J. A. (2018). The phosphoinositide phosphatase Sac1 regulates cell shape and microtubule stability in the developing Drosophila eye. Development 145(11). PubMed ID: 29752385

Forrest, S., Chai, A., Sanhueza, M., Marescotti, M., Parry, K., Georgiev, A., Sahota, V., Mendez-Castro, R. and Pennetta, G. (2013). Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis. Hum Mol Genet 22(13): 2689-2704. PubMed ID: 23492670

Griffiths, N. W., Del Bel, L. M., Wilk, R. and Brill, J. A. (2020). Cellular homeostasis in the Drosophila retina requires the lipid phosphatase Sac1. Mol Biol Cell: mbcE20020161. PubMed ID: 32186963

Jiang, K., Liu, Y., Fan, J., Zhang, J., Li, X. A., Evers, B. M., Zhu, H. and Jia, J. (2016). PI(4)P promotes phosphorylation and conformational change of Smoothened through interaction with its C-terminal tail. PLoS Biol 14(2): e1002375. PubMed ID: 26863604

Lee, S., Kim, S., Nahm, M., Kim, E., Kim, T. I., Yoon, J. H. and Lee, S. (2011). The phosphoinositide phosphatase Sac1 is required for midline axon guidance. Mol Cells 32(5): 477-482. PubMed ID: 22042447

Mao, D., Lin, G., Tepe, B., Zuo, Z., Tan, K. L., Senturk, M., Zhang, S., Arenkiel, B. R., Sardiello, M. and Bellen, H. J. (2019). VAMP associated proteins are required for autophagic and lysosomal degradation by promoting a PtdIns4P-mediated endosomal pathway. Autophagy 15(7): 1214-1233. PubMed ID: 30741620

Moustaqim-Barrette, A., Lin, Y. Q., Pradhan, S., Neely, G. G., Bellen, H. J. and Tsuda, H. (2014). The amyotrophic lateral sclerosis 8 protein, VAP, is required for ER protein quality control. Hum Mol Genet 23(8): 1975-1989. PubMed ID: 24271015

Pennetta, G., Hiesinger, P. R., Fabian-Fine, R., Meinertzhagen, I. A. and Bellen, H. J. (2002). Drosophila VAP-33A directs bouton formation at neuromuscular junctions in a dosage-dependent manner. Neuron 35(2): 291-306. PubMed ID: 12160747

Venditti, R., Masone, M. C., Rega, L. R., Di Tullio, G., Santoro, M., Polishchuk, E., Serrano, I. C., Olkkonen, V. M., Harada, A., Medina, D. L., La Montagna, R. and De Matteis, M. A. (2019). The activity of Sac1 across ER-TGN contact sites requires the four-phosphate-adaptor-protein-1. J Cell Biol 218(3): 783-797. PubMed ID: 30659099

Wallroth, A. and Haucke, V. (2018). Phosphoinositide conversion in endocytosis and the endolysosomal system. J Biol Chem 293(5): 1526-1535. PubMed ID: 29282290

Wei, H. C., Sanny, J., Shu, H., Baillie, D. L., Brill, J. A., Price, J. V. and Harden, N. (2003a). The Sac1 lipid phosphatase regulates cell shape change and the JNK cascade during dorsal closure in Drosophila. Curr Biol 13(21): 1882-1887. PubMed ID: 14588244

Wei, H. C., Shu, H. and Price, J. V. (2003b). Functional genomic analysis of the 61D-61F region of the third chromosome of Drosophila melanogaster. Genome 46(6): 1049-1058. PubMed ID: 14663523

Yavari, A., Nagaraj, R., Owusu-Ansah, E., Folick, A., Ngo, K., Hillman, T., Call, G., Rohatgi, R., Scott, M. P. and Banerjee, U. (2010). Role of lipid metabolism in smoothened derepression in hedgehog signaling. Dev Cell 19(1): 54-65. PubMed ID: 20643350

Biological Overview

date revised: 17 August 2020

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