InteractiveFly: GeneBrief

clueless: Biological Overview | References


Gene name - clueless

Synonyms -

Cytological map position - 52F5-52F7

Function - promotes damaged mitochondrial clearance

Keywords - required for normal mitochondrial function, negative regulator of the PINK1-Park pathway, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, works to promote damaged mitochondrial clearance

Symbol - clu

FlyBase ID: FBgn0034087

Genetic map position - chr2R:16,186,584-16,194,277

Classification - CLU domain (CLUstered mitochondria), Tetratricopeptide repeat

Cellular location - cytoplasmic, located at the mitochondrial outer membrane



NCBI links: Precomputed BLAST | EntrezGene
BIOLOGICAL OVERVIEW

Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1) and Parkin (Park), either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. This study shows that Drosophila Clueless (Clu), a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM) 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previous studies have found that clu genetically interacts with park in Drosophila female germ cells. This study shows that clu also genetically interacts with PINK1, and epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control (Sen, 2015).

Mitochondrial function is intimately linked to cellular health. These organelles provide the majority of ATP for the cell in addition to being the sites for major metabolic pathways such as fatty acid β-oxidation and heme biosynthesis. In addition, mitochondria are crucial for apoptosis, and they can irreparably damage the cell via oxidation when their biochemistry is abnormally altered. Given these many roles, tissues and cell types with high energy demands, such as neurons, are particularly sensitive to changes in mitochondrial function. This is also true for germ cell mitochondria because mitochondria are inherited maternally from the egg's cytoplasm and are thus the sole source of energy for the newly developing embryo (Sen, 2015).

Mitochondrial biology is complex owing to the dynamic nature of the organelle and the fact that most of the proteins required for function are encoded in the nucleus. In addition to the metabolites they provide, mitochondria undergo regulated fission, fusion and transport along microtubules. Because mitochondria cannot be made de novo, and tend to accumulate oxidative damage due to their biochemistry, they are subject to organelle and protein quality-control measures that involve mitochondrial and cytoplasmic proteases, as well as a specialized organelle-specific autophagy called mitophagy. However, the specific molecular signaling pathways between the nucleus and mitochondria that are used to sense which individual mitochondria are damaged during normal cellular homeostasis in vivo are not well understood. This study used the Drosophila ovary to identify genes regulating mitochondrial function and have characterized mitochondrial dynamics during Drosophila oogenesis. Germ cells contain large numbers of mitochondria that can be visualized at the single organelle level, making this system useful for studying genes that control mitochondrial function (Sen, 2015).

The gene clueless (clu) is crucial for mitochondrial localization in germ cells. Clu has homologs in many different species, and shows 53% amino acid identity to the human homolog, CLUH. The molecular role of Clu is not known. The yeast homolog, Clu1p, was found to interact with the eukaryotic initiation factor 3 (eIF3) complex in yeast and bind mRNA; however, the significance of this is not clear. CLUH has also been shown to bind mRNA. Flies mutant for clu are weak, uncoordinated, short-lived, and male and female sterile (Cox, 2009). Lack of Clu causes a sharp decrease in ATP, increased mitochondrial oxidative damage and changes in mitochondrial ultrastructure (Cox, 2009; Sen, 2013). Levels of Clu protein are homogeneously high in the cytoplasm and it is also found in large mitochondrially-associated particles. Although Clu clearly has an effect on mitochondria function, whether this is direct or indirect has not yet been established (Sen, 2015).

Parkin (Park), an E3 ubiquitin ligase, acts with PTEN-induced putative kinase 1 (PINK1) to target mitochondria for mitophagy. clu genetically interacts with park, and Clu particles are absent in park mutants, indicating that Clu might play a role in Park's mechanism (Cox, 2009; Sen, 2013). park and PINK1 have been identified as genes that, when mutated, cause early-onset forms of Parkinson's disease. Upon mitochondrial depolarization, PINK1 is stabilized on the mitochondrial outer membrane, recruiting Park, which then goes on to ubiquitinate many surface proteins, thus marking and targeting that mitochondrion for mitophagy. Before their biochemical interaction was recognized, PINK1 was placed upstream of park in a genetic pathway in Drosophila. Understanding Park and PINK1's role in mitochondrial quality control has shed light on the neurodegeneration underlying Parkinson's disease (Sen, 2015).

This study shows that Clu's mitochondrial role is well conserved, because the human homolog, CLUH, can rescue the fly mutant. Clu peripherally associates with mitochondria because it forms a complex with the mitochondrial outer-membrane proteins Porin and Translocase of the outer membrane (TOM) 20, supporting that the loss of mitochondrial function caused by lack of Clu is a direct effect. In addition, this study found that clu genetically interacts with PINK1 and, using epistasis, clu was placed upstream of park, but downstream of PINK1. Clu forms a complex with PINK1, and is able to interact with Park after the mitochondrial membrane potential is disrupted. Finally, lack of Clu causes PINK1 and Park to interact with each other, as well as causing a decrease in mitochondrial proteins, which suggests that Clu negatively regulates PINK1-Park function. Taken together, these data identify Clu as a mitochondrially-associated protein that plays a direct role in maintaining mitochondrial function and that binds TOM20, and support a role for Clu linking mitochondrial function to the PINK1-Park pathway (Sen, 2015).

Drosophila Clu is a large, highly conserved protein that shares its Clu and tetratricopeptide repeat (TPR) domains with its human homolog, CLUH. Expressing CLUH in flies that are mutant for clu rescues the mutant phenotypes; thus, the human protein can use the fly machinery to fulfill the role of Clu. To date, all the evidence supports the idea that Clu has a role in mitochondrial function; however, it has been unclear how direct it is. In this study, using IPs showed that Clu can associate with three proteins located on the mitochondrial outer membrane, TOM20, Porin and PINK1. Thus, Clu is not only a cytoplasmic protein, but can also be a peripherally associated mitochondrial protein, supporting the idea that this highly conserved protein directly affects mitochondrial function (Sen, 2015).

clu mutants share many phenotypes with park and PINK1 mutant flies, including flight muscle defects and sterility. Mitochondria are also mislocalized in PINK1 mutant germ cells, similarly to park mutants, and form large knotted clumps that include circularized mitochondria, which is consistent with increased fusion events. Mitochondria in clu mutant germ cells, on the other hand, do not show any signs of changes in fission or fusion (Cox, 2009). clu also genetically interacts with PINK1 and park, with double heterozygotes having clumped mitochondria in germ cells and a loss of Clu particles, and double knockdown of clu with PINK1 or park in flight muscle causing an increase in abnormal wing posture (Cox, 2009). Park functions in a pathway with PINK1 to elicit a mitophagic response, and overexpressing park can rescue PINK1 phenotypes in Drosophila. Using S2R+ cells and clu RNAi knockdown, this study found that overexpressing Park, but not PINK1, causes mitochondria to disperse. In adult flies, overexpressing full-length clu rescues the abnormal wing phenotype as well as mitochondrial phenotypes of PINK1 mutants, and overexpressing full-length clu or CLUH in PINK1, but not park, mutants rescues their thoracic indentation. These results place clu upstream of park, but downstream of PINK1. PINK1 stabilization on the mitochondrial outer membrane signals for Park to translocate to the organelle and subsequently ubiquitinate different proteins on the mitochondrial surface. Thus, it is somewhat surprising in Drosophila that loss of PINK1 can be rescued by increased amounts of Park, and suggests that there might be additional roles that Park plays in the cell. The data support the idea that an excess of Park overcomes deficits in mitochondrial function because it can rescue a loss of Clu as well. Mitochondrial clumping seems to be one of the responses to mitochondrial damage, in this system and in human tissue culture cells; thus, the dispersal upon Park overexpression in clu-RNAi-treated S2R+ cells is likely a sign of better mitochondrial health (Sen, 2015).

This study shows that Clu reciprocally immunoprecipitates with overexpressed PINK1 under normal cell culture conditions. PINK1 has been shown to directly bind TOM20, and Clu can also form a complex with TOM20, suggesting that all three proteins are found in close proximity at the mitochondrial membrane. Clu still immunoprecipitates with PINK1 when PINK1 is no longer targeted to the mitochondrial outer membrane (PINK1ΔMTS). This result indicates that Clu forms a complex with PINK1 independent of TOM20 or any other mitochondrial outer membrane proteins. Under normal conditions, PINK1 degradation happens so quickly that there are undetectable levels found at the outer mitochondrial membrane. Therefore, how is it possible that Clu is found in a complex with PINK1 in the absence of mitochondrial damage? It is likely that overexpressed PINK1 overwhelms the normal degradation process, thus becoming aberrantly stabilized at the outer mitochondrial membrane. Alternatively, it is possible that low levels of mitochondrial damage could account for the PINK1 being stabilized at the outer membrane, and then being able to interact with Clu (Sen, 2015).

Mitophagy ultimately leads to mitochondrial degradation in the lysosome. Currently, the literature involving Park and PINK1 uses mitochondrial protein levels as a read-out of mitophagy. However, recent data shows that different mitochondrial proteins have different half-lives, likely depending on what type of protein quality-control mechanism they use. Recent papers have examined protein half-life and found that Drosophila and yeast mitochondrial proteins, particularly those of Complex I in the case of flies, have increased half-lives when mitophagy proteins are missing. In addition, mitochondrial protein quality control does not always require destruction of the entire mitochondrion, but can selectively destroy certain proteins. For the mitochondrial proteins examined, all were greatly reduced in clu and PINK1 mutants, but not substantially altered in park mutants. This suggests that the turnover of the mitochondrial proteinsexamined is more sensitive to the absence of clu and PINK1 than park. This study found that Park and PINK1 form a complex in the absence of Clu. Thus, Clu is not necessary for this interaction, and loss of Clu causes a PINK1-Park interaction. This, plus the fact that Clu can be found at the outer mitochondrial membrane in a complex with both PINK1 and Park, suggests that Clu can influence mitochondrial quality or function, perhaps by regulating mitochondrial protein levels (Sen, 2015).

Yeast Clu1p was identified as a component of the eukaryotic initiation factor 3 (eIF3) complex and as an mRNA-binding protein. From IP and mass spectrometry data of the current study, there evidence that Clu can associate with the ribosome as well. Although CCCP is commonly used to force mitophagy and mitochondrial protein turnover, this treatment might not mimic the more subtle damage and changes mitochondria likely face in vivo. Mitochondrial protein import, for example, requires an intact mitochondrial membrane potential. Given the curent data, it is possible that Clu could function in co-translational import of proteins, as well as act as a sensor to couple PINK1-Park complex activation to how well protein import occurs. This would help explain why this study found that loss of Clu triggers a PINK1-Park interaction. In addition, Park and PINK1 directly interact with Porin and TOM20, respectively, placing them and Clu at the same place at the outer mitochondrial membrane. Recently, CLUH has been found to bind mRNAs for nuclear-encoded mitochondrial proteins, supporting a potential role in co-translational import. Further experiments are required to understand the precise relationship between Clu, TOM20, PINK1 and Park (Sen, 2015).

Mitochondria clearly undergo targeted destruction and require robust quality-control mechanisms, which are very active areas of investigation. PINK1 and Park's molecular mechanisms are particularly relevant to Parkinson's disease, given that inherited mutations in PARK2 and PINK1 can cause early-onset Parkinsonism. The molecular mechanisms that control mitophagy are becoming increasingly complex, involving membrane and cell biology; however, to date, the field has yet to visualize and understand the role of basal mitophagy levels in vivo. In the future, studying mitochondria and Clu function in Drosophila germ cells could lead to a better understand the role of mitochondrial protein turnover and quality control in the normal life cycle of tissues (Sen, 2015).

Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane

Mitochondrial function is tied to the nucleus, in that hundreds of proteins encoded by nuclear genes must be imported into mitochondria. While post-translational import is fairly well understood, emerging evidence supports that mitochondrial site-specific import, or co-translational import, also occurs. However, the mechanism and the extent to which it is used are not fully understood. Previous studies have shown that Clueless (Clu), a conserved multi-domain protein, associates with mitochondrial outer membrane proteins, including Translocase of outer membrane 20, and genetically and physically interacts with the PINK1-Parkin pathway. The human ortholog of Clu, Cluh, was shown to bind nuclear-encoded mitochondrially destined mRNAs. This study identified the conserved tetratricopeptide domain of Clu as predominantly responsible for binding mRNA. In addition, Clu was shown to interact with the ribosome at the mitochondrial outer membrane. Taken together, these data support a model whereby Clu binds to and mitochondrially targets mRNAs to facilitate mRNA localization to the outer mitochondrial membrane, potentially for site-specific or co-translational import. This role may link the presence of efficient mitochondrial protein import to mitochondrial quality control through the PINK1-Parkin pathway (Sen, 2016).

Clu is a large protein (~160 kDa) that contains several domains. With the exception of the N-terminal ms domain, each domain when deleted fails to rescue the mitochondrial clumping phenotype in S2R+ cells, as well as clu mutant phenotypes in vivo. Given that Clu is able to associate with proteins involved in multiple different processes (ribosomal proteins, mitochondrial outer membrane proteins, and the PINK1-Parkin complex), Clu may act as a type of scaffold that can bring together several mechanisms in order to maintain mitochondrial function and output. Previous work has shown lack of Clu causes an increase in mitochondrial oxidative damage and a decrease in ATP (Sen, 2013). Over-expressing single-domain deletions significantly reduced ATP levels below clu mutants alone. This could be because too much Clu can have a dominant negative effect by titrating out critical binding partners, thus negatively impacting mitochondrial function. Alternatively, high Clu levels may be toxic in and of themselves, however, overexpressing FL-clu does not appear to be toxic to flies (Sen, 2016).

While RNAi knockdown of Clu in S2R+ cells causes mitochondrial clumping, the cells themselves do not have reduced levels of ATP compared to control. This may be due to the differences in metabolism between cultured cells and cells in vivo . For example, there was a marked difference between the sedimentation profiles of Clu and Cluh. Cluh is found in the lighter fractions in extract from HeLa cells, whereas Clu from ovary extract was always found in the heaviest fractions in a sucrose gradient. HeLa cells, as well as many other transformed cultured cells, are known to use high amounts of glucose that appears to be used for glycolysis. For tumors, this is known as the Warburg effect. In addition, glucose has been documented to inhibit oxidative phosphorylation, which is known as the Crabtree effect. In fact, the culture media often used, DMEM, contains pre-diabetic levels of glucose, thus providing the cells with high concentrations of glucose. The different metabolism and use of mitochondria could be one explanation for the discrepancy between the sedimentation of Clu and Cluh (Sen, 2016).

Previously, Gao et al. showed that Cluh co-sediments with light membranes and the ribosomal fraction and is released with high salt, suggesting it is a ribosome associated protein (Gao, 2014). This analysis has been extended to show that Clu reciprocally co-IPs with three ribosomal proteins (RpLs) and two RpSs (small ribosomal proteins), as well as two eIF3 proteins, and thus does indeed bind the ribosome. Furthermore, addition of EDTA, which dissociates ribosomes, caused Clu to shift from the heaviest to lighter fractions in a sucrose gradient. However, most importantly, Clu is able to bind RpL7a in the mitochondrial fraction but not in the cytoplasm. While there are high levels of homogeneous Clu in the cytoplasm, Clu particles in Drosophila germ cells are clearly juxtaposed to mitochondria but do not co-localize (Cox, 2009; Sen, 2015). As was previously shown, Clu associates with mitochondrial outer membrane proteins (Sen, 2015). These data taken together support that Clu predominantly binds the ribosome at the outer mitochondrial membrane in vivo. In addition, Clu associates with TOM20, thus placing Clu in the same location at which mitochondrial protein import occurs (Sen, 2015; Sen, 2016 and references therein).

Drosophila third instar neuroblasts divide frequently (approximately every twenty five minutes) to ultimately create the adult brain. Components of the Myc pathway, which is responsible for ribosome biogenesis, have been shown to have differential expression in neuroblasts. The nucleolar protein Mushroom body miniature (Mbm), which is highly expressed in neuroblasts, is a transcriptional target of Myc and is involved in ribosome biogenesis (Hovhanyan, 2014). Conversely, Brain tumor (Brat) has been shown to be a negative regulator of Myc and becomes asymmetrically localized away from the neuroblast into the daughter cell during cell division. Work involving transcriptome analysis found that genes involved in ribosome biogenesis, as well as other processes, are highly represented in neuroblasts compared to differentiated neurons. Given that an increase in ribosome biogenesis appears intrinsically important for neuroblasts, it follows that ribosomal proteins should also be enriched in neuroblasts. This work shows that RpLs, as well as eIF3-S9, are highly enriched in neuroblasts (Sen, 2016).

Clu1p and Cluh are ribonucleoproteins, however which Clu domain binds mRNA has not been identified (Gao, 2014; Mitchell, 2013). This study shows the TPR domain of Clu is critical for mRNA binding. TPR domains are well known to facilitate protein-protein interactions. Proteins containing the related HAT domain (half a TPR) and pentatricopeptide repeats (PPRs) have been shown to directly bind RNA. HAT and PPR domains have similar sequence and structure to TPRs. Since UV-cross linking causes covalent bonds between nucleic acid and protein, the current data showing deletion of the TPR greatly decreases the amount of bound mRNA is consistent with it potentially directly binding mRNA (Sen, 2016).

These data strongly support that Clu normally binds to mRNA in vivo. Combined with data showing Clu binds ribosomal proteins, and can do so in the mitochondrial fraction, suggests that the role of Clu in the cell is to facilitate mRNA binding and association with the ribosome at the mitochondrial outer membrane. In fact, mitochondrial protein levels are globally reduced in Drosophila clu mutants, and specific mitochondrial proteins encoded by Cluh-bound mRNAs are reduced in cluh knockout immortalized mouse embryonic fibroblasts (Gao, 2014; Sen, 2015). Thus, Clu may act to provide site-specific translation or co-translational import of mitochondrial proteins. The molecular mechanism of co-translational import is well-established for endoplasmic reticulum bound proteins. It is increasingly clear that co-translational import occurs on mitochondria, however the mechanism is not as clear. Clu forms a complex with the mitophagy proteins PINK1 and Parkin, and PINK1 and TOM20 have been implicated in localized translation of mRNAs encoding respiratory chain proteins. It is possible that Clu may function to link lack of mitochondrial import and activity with mitochondrial damage sensing and destruction (Sen, 2015; Sen, 2016).

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).

Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage

Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, this study has identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. While mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, this study shows that overall brain morphology appears to be normal. This is explained by the observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus the Clu role in mitochondrial function is not critical during larval development, but is important for pupae and adults (Sen, 2013).

Clueless regulates aPKC activity and promotes self-renewal cell fate in Drosophila lgl mutant larval brains

Asymmetric cell division of Drosophila neural stem cells or neuroblasts is an important process which gives rise to two different daughter cells, one of which is the stem cell itself and the other, a committed or differentiated daughter cell. During neuroblast asymmetric division, atypical Protein Kinase C (aPKC) activity is tightly regulated; aberrant levels of activity could result in tumorigenesis in third instar larval brain. This study identified clueless (clu), a genetic interactor of parkin (park), as a novel regulator of aPKC activity. It preferentially binds to the aPKC/Bazooka/Partition Defective 6 complex and stabilizes aPKC levels. In clu mutants, Miranda (Mira) and Numb are mislocalized in small percentages of dividing neuroblasts. Adult mutants are short-lived with severe locomotion defects. Clu promotes tumorigenesis caused by loss of function of lethal(2) giant larvae (lgl) in the larval brain. Removal of clu in lgl mutants rescues Mira and Numb mislocalization and restores the enlarged brain size. Western blot analyses indicate that the rescue is due to the down-regulation of aPKC levels in the lgl clu double mutant. Interestingly, the phenotype of the park mutant, which causes Parkinson's Disease-like symptoms in adult flies, is reminiscent of that of clu in neuroblast asymmetric division. This study provides the first clue for the potential missing pathological link between temporally separated neurogenesis and neurodegeneration events; the minor defects during early neurogenesis could be a susceptible factor contributing to neurodegenerative diseases at later stages of life (Goh, 2013).

In this study, Clu, a protein expressed in L3 brain NBs, was found to be involved in regulating aPKC levels. In the absence of Clu, both Mira and Numb were delocalized in small percentages of mitotic NBs. Clu preferably binds to aPKC and Baz but not Lgl. In addition, this study also showed that Clu promotes a tumorigenesis phenotype in the lgl mutant. In the absence of Lgl, the function of Clu to maintain aPKC levels was sensitized. Drastic rescue was seen when clu was removed from the lgl mutant. The Western blot analyses indicated that in the lgl clu double mutant, both aPKC and p-aPKC levels were reduced, which was responsible for the rescue of lgl tumorigenesis phenotype. These data are most consistent with a model in which Clu is a member of aPKC/Baz/Par6 complex and presumably functions to maintain its stability (Goh, 2013).

If Clu is indeed involved in the maintenance of the aPKC/Baz/Par6 active complex, why does deletion of clu alone fail to generate any drastic phenotype? Deletion of clu only caused a low percentage of Mira and Numb mislocalization. The L3 brain of clu did not form tumors and the mutant could even survive all the way to adulthood, albeit it could only survive for 3-7 days. The key to that question may lie in its genetic interactor, lgl (Goh, 2013).

In the Western Blot analyses of whole larval brain lysate and knockdown cell line lysate of clu, a decrease was observed in Lgl as compared with the control. This may suggest that in clu mutant, there might also be a lower level of the inactive aPKC/Lgl/Par6 inactive complex. It is possible that the lack of Clu not only exposes the aPKC/Baz/Par6 complex to perturbations such as dephosphorylation or degradation, it also lowers Lgl levels, which in turn results in less aPKC/Lgl/Par6 complex. As a consequence, more aPKC can be released and forms a complex with Baz to replenish the decreasing pool of active complex. Thus, Lgl is able to fulfill its role as a molecular buffer in the clu mutant, shifting the equilibrium toward the more active aPKC/Baz/Par6 complex and compensating Clu function (Goh, 2013).

The primary aim of asymmetric cell division in NBs is to localize cell fate determinants such as Pros, Brat and Numb to the basal cortex so that after cytokinesis, they would only be inherited by the GMC. Numb and Mira have been found to be phosphorylated by aPKC and this phosphorylation resulted in apical exclusion of the proteins. The optimal levels of phosphorylation of Numb depend heavily on the equilibrium of two complexes, the active aPKC/Baz/Par6 complex, and the inactive aPKC/Lgl/Par6. Mira phosphorylation may depend on a similar aPKC complex. Studies so far had pointed to Lgl as the major buffering molecule to maintain this equilibrium, together with other phosphatases like PP4 and PP2A modulating the equilibrium or activity of the two complexes. There should be, however, additional molecules that help maintain this equilibrium in the Drosophila NBs (Goh, 2013).

Clu exhibited preferential binding capacity to the aPKC/Baz/Par6 complex rather than aPKC/Lgl/Par6, suggesting its participation in the maintenance or regulation of the equilibrium between the two complexes. Under extreme conditions, such as in the lgl mutant, lack of Lgl not only shifts the equilibrium entirely to the aPKC/Baz/Par6 complex, resulting in hyper-phosphorylation and subsequent delocalization of Numb and Mira, but also sensitizes Clu function on stabilizing aPKC/Baz/Par6. When clu is further deleted in the lgl mutant, the aPKC/Baz/Par6 complex could be destabilized, hence resulting in a significant decrease in the levels of both aPKC and p-aPKC, which in turn rescues Numb and Mira localization phenotypes in dividing NBs and restores L3 brain sizes. Western Blot analyses of whole larval brain lysate and knockdown BG3C2 cell lines supported this hypothesis (Goh, 2013).

Both clu and park have been shown previously to cause mitochondrial defects in adults. An initial suspicion was that ATP levels in clu NBs might contribute to the defects of neuroblast asymmetric cell division, as well as the rescue phenotype in the lgl mutant. A recent paper indicated that localization of mitochondria did not affect levels of ATP or cause any oxidative damage in the 3L brain, although they observed abnormal clustering in the clu mutant (Sen, 2013). Measurements of ATP levels in clu, lgl and lgl clu mutant brains also did not indicate any differences among them or the wild type brains. Thus, it is concluded that the clu phenotype observed in this study is very likely to be independent of mitochondrial activity (Goh, 2013).

Parkinson's disease is a neurodegenerative disease of the central nervous system which occurs in aged individuals. In patients with Parkinson's disease, there is massive loss of dopaminergic neurons which results in impairment in movement. The Drosophila ortholog of park encodes an E3 ligase, and they were found to have defective flight muscles and loss of dopaminergic neurons in park null mutants (Goh, 2013).

This has directly linked genes involved in neurodegenerative disease, clu and park, with lgl, a gene that is involved in NB asymmetric division during early development. Since these three genes are expressed in the same cells and are involved in the same process during neurogenesis, it is speculated that early defects in neurogenesis, although weak, may become a susceptible factor contributing to neurodegenerative diseases such as Parkinson disease at a much late stage of life. More in-depth studies on the links among genes involved in two developmentally separated events, asymmetric division during early neurogenesis and neurodegeneration due to aging, need to be done before the final conclusion can be reached (Goh, 2013).

clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin

Parkinson's disease has been linked to altered mitochondrial function. Mutations in parkin (park), the Drosophila ortholog of a human gene that is responsible for many familial cases of Parkinson's disease, shorten life span, abolish fertility and disrupt mitochondrial structure. However, the role played by Park in mitochondrial function remains unclear. This study describe a novel Drosophila gene, clueless (clu), which encodes a highly conserved tetratricopeptide repeat protein that is related closely to the CluA protein of Dictyostelium, Clu1 of Saccharomyces cerevisiae and to similar proteins in diverse metazoan eukaryotes from Arabidopsis to humans. Like its orthologs, loss of Drosophila clu causes mitochondria to cluster within cells. Strong clu mutations resemble park mutations in their effects on mitochondrial function and the two genes interact genetically. Conversely, mitochondria in park homozygotes become highly clustered. It is proposed that Clu functions in a novel pathway that positions mitochondria within the cell based on their physiological state. Disruption of the Clu pathway may enhance oxidative damage, alter gene expression, cause mitochondria to cluster at microtubule plus ends, and lead eventually to mitochondrial failure (Cox, 2009).

Mitochondria are often positioned within cells by motor-dependent movement along microtubules. Clu protein appears to contact mitochondria, especially in the large Clu particles, many of which are located adjacent to microtubules. The current experiments suggest that clu-induced mitochondrial aggregates are caused by changes in microtubule-based mitochondrial transport. In both the Drosophila brain and ovary, mitochondria associate with transport complexes containing the adaptor protein Milt that are linked to both plus-end-directed motors such as Khc and minus-end-directed motors such as Dhc. During oogenesis, loss of Khc, or of one of the Milt isoforms that interacts with Khc, cause mitochondria to cluster in cellular regions that are rich in microtubule minus ends, presumably because countervailing movement toward plus ends has been lost. Strikingly, mitochondria in clu mutant ovaries accumulate at predicted sites of microtuble plus ends; these include the proximal region of GSCs. Thus, in some situations, clu mutations may act by interfering with minus-end-directed mitochondrial movement along microtubules. Whether Clu particles correspond to mitochondrial transport complexes that mediate these changes, or whether Clu acts indirectly on mitochondrial positioning could not be determined with certainty from these studies (Cox, 2009).

In other cells, such as nurse cells, clu mutations also cause pronounced mitochondrial clustering, but that clustering is not mimicked by mutations in genes encoding microtubule motor proteins or Milt. Other systems of mitochondrial localization may predominate in such cells but still be subject to regulation by the Clu pathway. For example, in plant cells, Clu was postulated to control mitochondrial localization by regulating the choice between microtubule-dependent and microfilament-dependent transport. In particular, the TPR domain of Clu was proposed to repress the interaction of mitochondria with microtubules by competing for binding with the TPR region of Kinesin light chain, thereby allowing transport along actin to predominate. The loss of this postulated interaction through clu mutation predicts that mitochondria would accumulate at microtubule minus ends in these cells. Although the default position where mitochondria accumulate in the absence of Clu might vary depending on the particular cell type and transport systems involved, the use of a Clu-dependent pathway to control mitochondrial subcellular location appears to be widespread (Cox, 2009).

The experiments show that mitochondria require Clu to maintain their subcellular location and structural integrity. In the presence of even a relatively mild clu mutation, transcripts from genes involved in mitochondrial function and in protection from oxidative damage are reduced. Consequently, it is proposed that the normal role of Clu is to function in a pathway that controls the location and activity of mitochondria within the cell. Frequently, mitochondria may need to move to a different subcellular location in order to maximize access to substrates or to mitigate damage from oxidative metabolism. For example, in neurons, microtubule-dependent mitochondrial transport is modulated based on the level of respiratory activity. Clu protein would participate in a pathway that senses the internal physiological state of individual mitochondria and transduces this information into homeostatic changes in their positions and metabolic activities. When the Clu pathway is impaired, mitochondria would not move or operate normally, and might consequently suffer damage. This scenario is consistent with the changes that were observed in the inner mitochondrial membranes of Clu mutants, with their reduced levels of mitochondrial enzymes and the observed changes in nuclear gene expression. The greater severity of clu mutations in Drosophila compared with Dictyostelium cluA and yeast clu1 might be because of an intrinsically greater requirement for dynamic mitochondrial positioning in the specialized cells of complex metazoans. This model may provide a rationale for the unexpected effects of microtubule inhibitors on mitochondrial function that have been reported recently (Cox, 2009).

A model illustrates how the clu and park pathway(s) might link microtubule-based mitochondrial transport, mitochondrial physiology and oxidative damage (See Model for clu and park function). Under normal conditions, the clu and park pathway(s) would sense the physiological state of mitochondria and activate appropriate levels of minus-end-directed mitochondrial movement along microtubules. For example, mitochondria that are low in respiratory substrates might move to cellular regions where these substrates are abundant. Mitochondria in need of repair might move close to the nucleus where appropriate repair genes would be induced. In conjunction with active plus-end-directed motors, the result would be that mitochondria move dynamically to locations throughout the cell that are appropriate to their physiological state. However, without functional clu and park pathway(s), or if the local level of toxic metabolic products exceeded a threshold, the mitochondria in question would cease minus-end-directed transport and undergo concerted plus-end-directed movement. In many cells, the major locus of microtubule minus ends is found near the nucleus so that plus-end-directed movement would increase the distance between reactive oxygen production and the nuclear DNA (Cox, 2009).

These studies have several implications for understanding Parkinson's disease. According to the model, the Clu pathway would contribute strongly to the ability of mitochondria to remain functional during aging, despite the high metabolic requirements and environmental stresses experienced by many tissues. This may be particularly important in neural cells such as those that are compromised in Parkinson's disease. In a cell whose Clu pathway is compromised, mitochondria would operate less efficiently, suffer more damage and wear out faster because they would spend more time in cellular locations that are not appropriate to their metabolic state. The number of mitochondria producing elevated levels of reactive oxygen species would rise, increasing reactive oxygen damage to the nucleus and other cellular components, thereby leading to greater cell death. Consequently, the level of Clu pathway function within an individual might influence their susceptibility to sporadic Parkinson's disease and to other late-onset neurological disorders (Cox, 2009).

Mitochondrial mutations may fall into two classes based on their phenotype and on their relationship to the Clu pathway. Some mutations, such as those affecting mitochondrial ribosomal proteins, cause bang sensitivity, but differ phenotypically from clu mutations. By contrast, mutations in park, pink1, rho7, mitochondrial ATP6, and mitochondrial cytochrome oxidase resemble clu mutations in causing general defects in movement, flight, muscle or nerve degeneration, male fertility, and longevity. The effects of this latter class of mutations may be too severe to be compensated for by Clu pathway operation, leading to mitochondrial mis-positioning and increased production of reactive oxygen species. Consistent with this, expression of anti-oxidants partially suppresses the effects of pink1 on neurodegeneration. Mutations in some of these genes, in addition to park, may also cause mitochondrial clustering and interact genetically with clu. Clearly, further studies of the Clu pathway will deepen understanding of how mitochondria are maintained in cells and why they sometimes become damaged with age (Cox, 2009).


Functions of Clueless orthologs in other species

CLUH regulates mitochondrial biogenesis by binding mRNAs of nuclear-encoded mitochondrial proteins

Mitochondrial function requires coordination of two genomes for protein biogenesis, efficient quality control mechanisms, and appropriate distribution of the organelles within the cell. How these mechanisms are integrated is currently not understood. Loss of the Clu1/CluA homologue (CLUH) gene led to clustering of the mitochondrial network by an unknown mechanism. This study found that CLUH is coregulated both with genes encoding mitochondrial proteins and with genes involved in ribosomal biogenesis and translation. Functional analysis identifies CLUH as a cytosolic messenger ribonucleic acid (RNA; mRNA)-binding protein. RNA immunoprecipitation experiments followed by next-generation sequencing demonstrated that CLUH specifically binds a subset of mRNAs encoding mitochondrial proteins. CLUH depletion decreased the levels of proteins translated by target transcripts and caused mitochondrial clustering. A fraction of CLUH colocalizes with tyrosinated tubulin and can be detected close to mitochondria, suggesting a role in regulating transport or translation of target transcripts close to mitochondria. These data unravel a novel mechanism linking mitochondrial biogenesis and distribution (Gao, 2014).


REFERENCES

Search PubMed for articles about Drosophila Clueless

Cox, R. T. and Spradling, A. C. (2009). Clueless, a conserved Drosophila gene required for mitochondrial subcellular localization, interacts genetically with parkin. Dis Model Mech 2: 490-499. PubMed ID: 19638420

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: 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: 353-364. PubMed ID: 23835532

Hovhanyan, A., Herter, E. K., Pfannstiel, J., Gallant, P. and Raabe, T. (2014). Drosophila mbm is a nucleolar myc and casein kinase 2 target required for ribosome biogenesis and cell growth of central brain neuroblasts. Mol Cell Biol 34: 1878-1891. PubMed ID: 24615015

Mitchell, S. F., Jain, S., She, M. and Parker, R. (2013). Global analysis of yeast mRNPs. Nat Struct Mol Biol 20: 127-133. PubMed ID: 23222640

Sen, A., Damm, V. T. and Cox, R. T. (2013). Drosophila clueless is highly expressed in larval neuroblasts, affects mitochondrial localization and suppresses mitochondrial oxidative damage. PLoS One 8: e54283. PubMed ID: 23342118

Sen, A., Kalvakuri, S., Bodmer, R. and Cox, R. T. (2015). Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila. Dis Model Mech 8: 577-589. PubMed ID: 26035866

Sen, A. and Cox, R. T. (2016). Clueless is a conserved ribonucleoprotein that binds the ribosome at the mitochondrial outer membrane. Biol Open [Epub ahead of print]. PubMed ID: 26834020

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


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date revised: 22 December 2017

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