InteractiveFly: GeneBrief

porin: Biological Overview | References

The eukaryotic porin, also called the Voltage Dependent Anion-selective Channel (VDAC), is the main pore-forming protein of the outer mitochondrial membrane. In Drosophila melanogaster, a cluster of genes evolutionarily linked to VDAC is present on chromosome 2L. The main VDAC isoform, called VDAC1 (Porin1), is expressed from the first gene of the cluster. The porin1 gene produces two splice variants, 1A-VDAC and 1B-VDAC, with the same coding sequence but different 5' untranslated regions (UTRs). The influence of the two 5' UTRs, 1A-5' UTR and 1B-5' UTR, was studied on transcription and translation of VDAC1 mRNAs. In porin-less yeast cells, transformation with a construct carrying 1A-VDAC results in the expression of the corresponding protein and in complementation of a defective cell phenotype, whereas the 1B-VDAC sequence actively represses VDAC expression. Identical results were obtained using constructs containing the two 5' UTRs upstream of the GFP reporter. A short region of 15 nucleotides in the 1B-5' UTR should be able to pair with an exposed helix of 18S ribosomal RNA (rRNA), and this interaction could be involved in the translational repression. These data suggest that contacts between the 5' UTR and 18S rRNA sequences could modulate the translation of Drosophila 1B-VDAC mRNA. The evolutionary significance of this finding is discussed (Leggio, 2018).

The voltage-dependent anion-selective channel (VDAC), also known as mitochondrial porin, is the most abundant protein found in the outer mitochondrial membrane of all eukaryotes. VDAC is the main gateway for the entry and exit of mitochondrial metabolites, and thus it is suspected to control energetic exchanges between the mitochondria and the rest of the cell. Porin/VDAC interacts with several kinases or structural proteins of the cytoskeleton, such as microtubule-associated protein, tubulin and the dynein light chain Tctex-1. Furthermore, porin/VDAC mediates the Ca2+ traffic in the mitochondria. The role of VDAC in cancer is well known. VDAC1 contributes to the phenotype of cancer cells, regulating cellular energy production and metabolism. Indeed, this protein is overexpressed in many cancer types, and silencing of VDAC1 expression induces an inhibition of tumour development. Among others, VDAC1 controls, together with other proteins, the release of the pro-apoptotic factors from mitochondria, e.g. cytochrome c. VDAC1 can also regulate mitochondria-mediated apoptosis by interacting with hexokinases I and II and with proteins of the Bcl2 family, some of which are also highly expressed in many cancers. Moreover, the involvement of VDAC1 in many neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson's, Huntington's25 and Alzheimer's, has also been widely proven (Leggio, 2018).

Lower and higher eukaryotic cells express different sets of porin. In the budding yeast, Saccharomyces cerevisiae, two different porin genes have been identified, POR1 and POR2. In higher eukaryotes, like the mouse and the human, three porin/VDAC proteins are expressed. The structures of mouse and human VDAC1 and that of zebrafish VDAC2 have been resolved and found to exhibit a folding pattern that is similar overall. In contrast, there is a general consensus that each of the VDAC isoforms has distinct physiological roles, because they could specifically interact with different proteins or be differentially sensitive to oxidation by reactive oxygen species (Leggio, 2018).

In Drosophila melanogaster, the genomic locus 2L 32B displays a cluster of four spatially close genes that are evolutionarily linked to VDAC35. These genes share the same exon-intron organization and are very likely to be the result of gene duplication events. However, the main known protein is the product of the first gene (De Pinto, 1989). The second gene in the sequence, porin2, was shown to be expressed in vivo (by histological stainings) and to be able to form permeable pores (using the recombinant protein) (Aiello, 2004). The porin1 gene, which produces VDAC1, is encoded by two main transcripts, 1A-VDAC1 and 1B-VDAC1. These transcripts show two alternative 5' untranslated regions (UTRs) (corresponding to alternative exons 1A or 1B) but the same protein-encoding open reading frame (ORF) and the same 3' UTR sequence ending at one of three different alternative polyadenylation sites. The ORF is for VDAC1, the pore-forming protein of the fly. Thus, in the fly, two alternative splice variants (transcripts) are expressed and are present at all fly development stages and in the same tissues, at the same time, but 1A-VDAC is more abundant (Oliva, 1998). VDAC1 from D. melanogaster has been purified, and its gene has been cloned, sequenced and mapped. This protein shows very conserved functional features (De Pinto, 1989), and it is indeed about 60% identical to the VDAC mammalian isoforms (Leggio, 2018).

The presence of alternative 5' UTRs raised interest and was further investigated. Drosophila transposable P elements, when inserted into the porin locus, abolished VDAC expression and were found to produce a lethal phenotype35. Imprecise excision of such P elements showed that deletion of exon 1B and of its flanking sequences apparently has no effect on normal fly development or on VDAC protein level, whereas deletion of exon 1A suppresses VDAC protein expression. It was therefore suggested that 1B-VDAC could be an unproductive transcript (Leggio, 2018).

This study investigated the function of the two alternative Drosophila 5' UTRs. Overall, the current results suggest that a specific mechanism could be involved in the 1B-VDAC translational regulation. The biological significance of this mechanism is discussed (Leggio, 2018).

This work focused on the regulation of expression of VDAC1 in D. melanogaster. In this species, the porin1 gene produces two alternative transcripts named 1A-VDAC and 1B-VDAC, containing an identical coding sequence but two completely different 5' UTRs. To gain further insights into the biological function of these two alternative splicing forms of VDAC, they were introduced into a VDAC-lacking system, an established S. cerevisiae strain where the porin1 gene was inactivated (δpor1 strain). The advantage of the yeast cell is its viability (under fermentative conditions), whereas D. melanogaster cells cannot survive the deletion of the VDAC1 gene (Leggio, 2018).

In δpor1 yeast, the heterologous 1A-5' UTR directed transcription and translation of VDAC and of GFP used as a reporter; in contrast, the 1B-5' UTR directed the transcription but not the translation of the VDAC or the reporter gene. These results confirm that only the 1A-VDAC, but not the 1B-VDAC, is able to complement the growth defect of the δpor1 yeast cells. Similar data were obtained in Drosophila cells by using a luciferase reporter gene downstream of the 1A- or 1B-5' UTR. These results suggest that the 1B-5' UTR affects VDAC expression by inhibiting protein translation. Furthermore, the results suggest that this mechanism is independent of the coding region cloned downstream of the 5'-UTR (Leggio, 2018).

This study aimed to understand the mechanism responsible for the negative influence of the 1B-5' UTR on the translation of the coding sequences fused downstream. Gene expression in eukaryotic cells is regulated at multiple levels, including mRNA translation. Such control allows rapid changes in protein concentrations and, thus, it is used to maintain cellular homeostasis. Most translation regulation is exerted at the very first stage, when the AUG start codon is identified after the 5' UTR ribosome scanning. Consequently, any occurrence that prevents or inhibits the ability of the ribosome to scan the 5' UTR reduces the efficiency of translation initiation. Mechanisms that produce this effect are well known. Therefore, some of these were assayed, such as the presence of uORFs or stable secondary structures and the association with regulatory RBPs (Leggio, 2018).

The possibility was ruled out that the small upstream ORF (uORF) located in the 1B sequence is involved in translational control. Bioinformatic analysis suggested that no putative strong secondary structure in the untranslated region of 1B-VDAC mRNA should be involved in the inhibition of translation. In addition, bioinformatic predictive analysis of RBPs showed that there is no known RBP specific for the 1B-5' UTR, within the limitations of computational tools. Moreover, the possible involvement of miRNAs was not considered, because the 3' UTR of 1B-VDAC is included in the corresponding 3' UTR of 1A-VDAC, which is longer. Therefore, because a regulatory mechanism involving a miRNA action targeted to this region of 1B-VDAC mRNA could not be specific for the 1B-mRNA, this mechanism was ruled out (Leggio, 2018).

Using a mutagenesis scanning approach, the 16-31 nucleotide region of the 1B-5' UTR sequence was identified as responsible in yeast for the inhibitory effect on translation. The defect in the growth of the δpor1 yeast strain was indeed complemented when the strain was transformed with 1B(Δ16-31)-VDAC mutant, underlining that its removal is sufficient to re-establish the translation. It was also verified that the 16-31 sequence works similarly in Drosophila, although the translation inhibition must rely also on others factors. Therefore, by MS analysis of the proteins bound to an RNA oligo containing the 10-34 sequence of the 1B-5' UTR, proteins were sought that were directly or indirectly involved in the translation control. In particular, eIF4A, eIF5a and Asc1 were recognized. eIF4A is a RNA helicase working in the first stage of translation as a subunit of the cap-binding complex eIF4F, which unwinds the RNA secondary structures in the 5' UTR. Asc1/RACK1 associates with the 40S subunit close to the mRNA exit channel, where it interacts with eIF4E of eIF4F51. Asc1/RACK1 is involved in the control of the translation of housekeeping genes and, in general, represses gene expression. It is known that RACK1 loss-of-function mutations cause early developmental lethality in the mouse and the fly, like VDAC knockout organisms. Moreover, in yeast, loss of ASC1 reduces translation of mitochondrial r-proteins and, like for lack of VDAC1, causes cells to be unable to use non-fermentable carbon sources, demonstrating a direct control of ASC1 on mitochondria functionality. Interestingly, RACK1 has many interaction partners, ranging from kinases and signalling proteins to membrane-bound receptors and ion channels. Thus, under stress conditions, RACK1 can function as a signalling hub of newly synthesised proteins (Leggio, 2018).

From this viewpoint, it can be hypothesised that in yeast the 16-31 sequence might prevents eIF4A function, maybe trapping eIF4A in an inactive conformation. In Drosophila, 1B-VDAC translation could be repressed at the starting point by the coordinated action of more molecules, probably recruited in situ by RACK1. Gus1, which together with Arc1, is known to form a protein complex operating in the control of translation, was identified. In addition, the presence of two different heat-shock proteins (Hsp12 and Hsp76) in this pool of interacting proteins should indicate their recruitment after stress conditions (Leggio, 2018).

The ability of the 1A- and 1B-5' UTR sequences to contact protein-free domains of 18S rRNA, the only rRNA in the 40S subunit, was also tested. Because 18S rRNA mutations impair the integrity of the scanning-competent pre-initiation complex and/or its joining together with the 60S subunit, the translation initiation rate might be reduced by strong and long-range interactions between the protein-free domains of 18S rRNA and the 5' UTR(s) of the incoming mRNA. It has already been demonstrated in eukaryotes that gene expression regulation at the level of translation may occur thanks to specific interactions between mRNAs and rRNA domains. In particular, a highly specific sequence complementarity between 18S rRNA and the 5' UTRs of mRNAs across species has been predicted; this complementarity may modulate the scanning processivity of the 40S subunit through the 5' UTR of mRNAs, which could even stall the initiating PICs in the case of long-range interactions (Leggio, 2018).

In particular, by prediction analysis of RNA:RNA interactions between yeast 18S rRNA and the two alternative D. melanogaster VDAC mRNAs (1A-VDAC and 1B-VDAC), it was found that, in yeast as in D. melanogaster, almost the whole 1B-5'UTR sequence is able to strongly interact with a long sequence of 18S rRNA. In contrast, the 1B(Δ16-31)-5'UTR sequence can only weakly interact with a short sequence of rRNA in the 40S subunit, thus showing a behaviour similar to that 1A-5' UTR. These results underline the relevance of 1B-5' UTR and, in particular in yeast, of its 16-31 sequence for the mechanism of translation control. Interestingly, it was also found that some regions of the rRNA sequence involved in the interaction with the 1B-5' UTR fold in solvent-exposed domains, and some of them are turned towards the mRNA path of the ribosome 40S subunit. Therefore, these rRNA domains should be able to contact the 5' UTR in the incoming 1B-VDAC mRNA, producing a stop in the ribosome scanning. It is noteworthy that a sequence of about 35 nucleotides can be allocated inside the ribosomal mRNA path of PIC and that it was found that almost the whole 1B-5' UTR sequence, (2-116 nucleotides), may potentially interact with three 18S rRNA helices (helix 35, helix 36 and a portion of the helix 34) arranged near the mRNA path at the neck of 40S. In addition, the large helix 33, together with parts of helix 31 and helix 32, being arranged at the beck of the 40S subunit, could easily interact with the 1B-5' UTR. In this way, the 1B-VDAC mRNA translation rate would be negatively controlled by its 5' UTR sequence through the collective action of several interactions with 18S rRNA, the result of which would be a strong delay in ribosome scanning of 1B-VDAC. Probably, this effect in Drosophila could also be the result of additional interactions with fly-specific proteins, ribosomal or not. In any case, it is extremely relevant that the sequences encompassing these rRNA helices are highly conserved between S. cerevisiae and D. melanogaster; this indicates that the mechanism described in the mixed yeast-fly system is likely to act in D. melanogaster (Leggio, 2018).

VDAC is an essential but dangerous protein. Its function as a pro-apoptotic factor is well known and therefore it is essential for the cell to implement a suitable control of VDAC protein level. Also, specific conditions of cell growth involving high energy demand are known to induce up-regulation of VDAC associated with the requirement of mitochondrial biogenesis. Furthermore, these events must be coordinated with the expression of the other mitochondrial proteins, codified by the nuclear genome and from mitochondrial DNA. Therefore, it is conceivable to suppose the presence in the cell of a 'sentry' molecule able to sense, directly or indirectly, the amount of this crucial protein. It was demonstrated that in Drosophila the level of 1B-VDAC transcript is highly increased as a result of overexpression of 1A-VDAC mRNA. When the level of the 1B-VDAC transcript was increased by its overexpression, the endogenous 1A-VDAC mRNA level was meaningfully reduced. Importantly, the results show that the unproductive 1B-VDAC mRNA is able to respond to 1A-VDAC transcript levels, and thus it might work as a molecule signalling the need for activation of mitochondrial biogenesis. This hypothetical role of 1B-VDAC mRNA is supported by its interaction with Asc1/RACK1. Asc1/RACK1 responds to multiple signals, and might act to coordinate the expression of other mitochondrial proteins and thus affect cell respiration (Leggio, 2018).

In addition, the assignment of this important role to 1B-VDAC mRNA might lead to an understanding of why the evolution of the Drosophila genus proceeded towards the acquisition of an alternative 5' UTR with specific features (Leggio, 2018).

In conclusion, these results extend earlier reports and provide further evidence that in D. melanogaster the 1A-VDAC transcript is responsible for protein expression, while the alternative 1B-VDAC mRNA is not active in this respect. Moreover, this work showed that a specific mechanism could be responsible for the translation inhibition of the alternative D. melanogaster 1B-VDAC1 transcript (Leggio, 2018).

Loss of porin function in dopaminergic neurons of Drosophila is suppressed by Buffy

Mitochondrial porin, also known as the voltage-dependent anion channel (VDAC), is a multi-functional channel protein that shuttles metabolites between the mitochondria and the cytosol and implicated in cellular life and death decisions. The inhibition of porin under the control of neuronal Ddc-Gal4 result in short lifespan and in an age-dependent loss in locomotor function, phenotypes that are strongly associated with Drosophila models of Parkinson disease. Loss of porin function was achieved through exploitation of RNAi while derivative lines were generated by homologous recombination and tested by PCR. The expression of human α-synuclein transgene in neuronal populations that include dopamine producing neurons under the control of Ddc-Gal4 produces a robust Parkinson disease model, and results in severely reduced lifespan and locomotor dysfunction. In addition, the porin-induced phenotypes are greatly suppressed when the pro-survival Bcl-2 homologue Buffy is overexpressed in these neurons and in the developing eye adding to the cellular advantages of altered expression of this anti-apoptotic gene. When α-synuclein was co-expressed along with porin, it results in a decrease in lifespan and impaired climbing ability. This enhancement of the α-synuclein-induced phenotypes observed in neurons was demonstrated in the neuron rich eye, where the simultaneous co-expression of porin-RNAi and α-synuclein resulted in an enhanced eye phenotype, marked by reduced number of ommatidia and increased disarray of the ommatidia. It is concluded that the inhibition of porin in dopaminergic neurons among others results in reduced lifespan and age-dependent loss in climbing ability, phenotypes that are suppressed by the overexpression of the sole pro-survival Bcl-2 homologue Buffy. The inhibition of porin phenocopies Parkinson disease phenotypes in Drosophila, while the overexpression of Buffy can counteract these phenotypes to improve the overall 'healthspan' of the organism (M'Angale, 2016).

Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex and Porin in Drosophila

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).

Age-dependent posttranslational modifications of voltage-dependent anion channel 1

The accumulation of oxidative damage in mitochondrial proteins, membranes and DNA during ageing is supposed to lead to mitochondrial inactivation, downstream molecular impairments and subsequent decline of biological systems. In a quantitative study investigating the age-related changes of mitochondrial proteins on the level of oxidative posttranslational modifications, a set of conserved biomarkers was found across ageing models in five species with consistent oxidative break-up of tryptophan residues and formation of N-formyl kynurenine. In an additional proteomic profiling of a long-living Drosophila mutant overexpressing mitochondrial Hsp22 and controls, age-related redundant isoforms were found of voltage-dependent anion channel 1 (VDAC-1). A re-examination of data from human umbilical vein endothelial cells (with normal and chemically accelerated in vitro ageing), revealed similar age-dependent alterations of voltage-dependent anion channel isoforms. Building on these results, the expression of VDAC-1 was examined in an in vitro model of excitotoxicity. Glutamate-induced calcium toxicity in neurons was found to induces changes of voltage-dependent anion channel 1 related to downstream events of mitochondrial apoptosis like poly-ADP-ribosylation (Groebe, 2010).

Drosophila Porin/VDAC affects mitochondrial morphology

Voltage-dependent anion channel (VDAC) has been suggested to be a mediator of mitochondrial-dependent cell death induced by Ca(2+) overload, oxidative stress and Bax-Bid activation. To confirm this hypothesis in vivo, Drosophila VDAC (porin) mutants wetr generated and characterized, and it was found that Porin is not required for mitochondrial apoptosis, which is consistent with the previous mouse studies. A novel physiological role of Porin was also discovered. Loss of porin resulted in locomotive defects and male sterility. Intriguingly, porin mutants exhibited elongated mitochondria in indirect flight muscle, whereas Porin overexpression produced fragmented mitochondria. Through genetic analysis with the components of mitochondrial fission and fusion, this study found that the elongated mitochondria phenotype in porin mutants were suppressed by increased mitochondrial fission, but enhanced by increased mitochondrial fusion. Furthermore, increased mitochondrial fission by Drp1 expression suppressed the flight defects in the porin mutants. Collectively, this study showed that loss of Drosophila Porin results in mitochondrial morphological defects and suggested that the defective mitochondrial function by Porin deficiency affects the mitochondrial remodeling process (Park, 2010).

Neurologic dysfunction and male infertility in Drosophila porin mutants: a new model for mitochondrial dysfunction and disease

Voltage-dependent anion channels (VDACs) are a family of small pore-forming proteins of the mitochondrial outer membrane found in all eukaryotes. VDACs play an important role in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, and three distinct mammalian isoforms have been identified. Animal and cell culture experiments suggest that the various isoforms act in disparate roles such as apoptosis, synaptic plasticity, learning, muscle bioenergetics, and reproduction. In Drosophila melanogaster, porin is the ubiquitously expressed VDAC isoform. Through imprecise excision of a P element insertion in the porin locus, a series of hypomorphic alleles have been isolated, and analyses of flies homozygous for these mutant alleles reveal phenotypes remarkably reminiscent of mouse VDAC mutants. These include partial lethality, defects of mitochondrial respiration, abnormal muscle mitochondrial morphology, synaptic dysfunction, and male infertility, which are features often observed in human mitochondrial disorders. Furthermore, the observed synaptic dysfunction at the neuromuscular junction in porin mutants is associated with a paucity of mitochondria in presynaptic termini. The similarity of VDAC mutant phenotypes in the fly and mouse clearly indicate a fundamental conservation of VDAC function. The establishment and validation of a new in vivo model for VDAC function in Drosophila should provide a valuable tool for further genetic dissection of VDAC role(s) in mitochondrial biology and disease, and as a model of mitochondrial disorders potentially amenable to the development of treatment strategies (Graham, 2010).

Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane that are found in all eukaryotes. VDACs are thought to play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, in overall energy metabolism via interactions with cytosolic kinases, and a debated role in programmed cell death (apoptosis). The mammalian genome contains three VDAC loci termed Vdac1, Vdac2, and Vdac3, raising the question as to what function each isoform may be performing. Based upon expression studies of the mouse VDACs in yeast, biophysical differences can be identified but the physiologic significance of these differences remains unclear. Creation of 'knockout' cell lines and mice that lack one or more VDAC isoforms has led to the characterization of distinct phenotypes that provide a different set of insights into function which must be interpreted in the context of complex physiologic systems. Functions in male reproduction, the central nervous system and glucose homeostasis have been identified and require a deeper and more mechanistic examination. Annotation of the genome sequence of Drosophila melanogaster has recently revealed three additional genes (CG17137, CG17139, CG17140) with homology to porin, the previously described gene that encodes the VDAC of D. melanogaster. Molecular analysis of these novel VDACs has revealed a complex pattern of gene organization and expression. Sequence comparisons with other insect VDAC homologs suggest that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. Striking similarities to mouse VDAC mutants can be found that emphasize the conservation of function over a long evolutionary time frame (Craigen, 2008).

Effects of a mutation in the Drosophila porin gene encoding mitochondrial voltage-dependent anion channel protein on phototransduction

Mitochondrial porins, also know as VDACs (voltage-dependent anion channels), play an important role in regulating energy metabolism, apoptosis, and the transport of metabolites across the mitochondrial outer membrane. So far three distinct isoforms of VDAC (VDAC1-3) have been reported in vertebrates, but their functions remain unknown. The annotation database of the Drosophila melanogaster genome sequence has identified four genes (porin, CG17137, CG17139, and CG17140) encoding different isoforms of VDACs. Post-translational modifications of PORIN were identified that are specific to D. melanogaster eyes. The P-element insertion in the porin gene, porin(G2294), was identifed that is homozygous viable whereas all the porin mutants previously reported are homozygous lethal at the pupal stage. The mutant does not show any defects in fly morphology, survival, and photoreceptor structure. The mutant, however, produces <10% of the normal level of wild-type (WT) porin transcripts and 16.5% of WT level of the PORIN protein. The P-element insertion affects only the expression of Class I transcript but not Class II transcript of the porin gene. Unlike in WT, the mutant displays an ERG (electroretinogram) that is not maintained during a prolonged light stimulus. The revertant obtained from remobilization of the P-element in the mutant produces the WT level of porin transcripts and PORIN protein, and shows a normal ERG response. These data suggest that the PORIN protein is important in maintaining a photoreceptor response during prolonged stimulation (Lee, 2007).

Functional characterization of a second porin isoform in Drosophila melanogaster

Mitochondrial porins or voltage-dependent anion-selective channels are channel-forming proteins mainly found in the mitochondrial outer membrane. Genome sequencing of the fruit fly Drosophila melanogaster revealed the presence of three additional porin-like genes. No functional information was available for the different gene products. This work has studied the function of the gene product closest to the known Porin gene (CG17137 coding for DmPorin2). Its coding sequence was expressed in Escherichia coli. The recombinant DmPorin2 protein is able to form channels similar to those formed by DmPorin1 reconstituted in artificial membranes. Furthermore, DmPorin2 is clearly voltage-independent and cation-selective, whereas its counterpart isoform 1 is voltage-dependent and anion-selective. Sequence comparison of the two porin isoforms indicates the exchange of four lysines in DmPorin1 for four glutamic acids in DmPorin2. Two of them (Glu-66 and Glu-163) were mutated to lysines to investigate their role in the functional features of the pore. The mutants E163K and E66K/E163K are endowed with an almost full inversion of the ion selectivity. Both single mutations partially restore the voltage dependence of the pore. An additional effect found with the double mutant E66K/E163K was the restoration of voltage dependence. Protein structure predictions highlight a 16 beta-strand pattern, typical for porins. In a three-dimensional model of DmPorin2, Glu-66 and Glu-163 are close to the rim of the channel, on two opposite sides. DmPorin2 is expressed in all the fly tissues and in all the developmental stages tested. The main conclusions are as follows. 1) The CG17137 gene may express a porin with a functional role in D. melanogaster. 2) Two amino acids were identified of major relevance for the voltage dependence of the porin pore (Aiello, 2004).

Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse

A cDNA encoding mitochondrial porin has been identied in Drosophila melanogaster, and its chromosomal localization was shown. Such cDNA was used as a probe for screening a genomic library. A 4494-bp genomic region was cloned and sequenced that contained the whole gene for the mitochondrial porin or VDAC. D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3' untranslated sequence (3'-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5'-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5'-UTR sequence, demonstrated to be transcribed by 5'-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2-1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites (Oliva, 1998).

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster

Polyclonal antibodies have been raised against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. This study presents the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family (Messina, 1996).

Characterization of the mitochondrial porin from Drosophila melanogaster

Mitochondrial porin was isolated from the fruit fly Drosophila melanogaster at different developmental stages, starting from whole mitochondria. The porin from adults' mitochondria was fully characterized. The protein had a molecular mass of 31 kDa as judged from sodium dodecylsulfate electrophoretograms. It was very resistive against digestion with V8 proteinase of Staphylococcus aureus and a larger number of fragments were only obtained after digestion with papain. Drosophila porin showed little interaction with antibodies raised against mitochondrial porins from mammalia and Neurospora crassa, but a strong reactivity with antibodies raised against yeast porin. Reconstitution experiments with planar lipid bilayer membranes showed that the protein was able to form ion-permeable pores with a single-channel conductance of 0.41 nS in 0.1 M KCl. At low transmembrane voltages Drosophila porin had the properties of a general diffusion pore with an estimated effective diameter of about 1.7 nm and a small selectivity for anions over cations. Voltages larger than 20 to 30 mV resulted in a closure of the pore. The closed states of the pore were found to be cation-selective. The addition of a synthetic polyanion to the aqueous phase on one side of the membrane resulted in an asymmetric shift of the voltage dependence and the pore became already closed at very small voltages negative at the cis-side (the side of the addition of the polyanion) (De Pinto, 1989).

Search PubMed for articles about Drosophila Porin

Aiello, R., Messina, A., Schiffler, B., Benz, R., Tasco, G., Casadio, R. and De Pinto, V. (2004). Functional characterization of a second porin isoform in Drosophila melanogaster. DmPorin2 forms voltage-independent cation-selective pores. J Biol Chem 279(24): 25364-25373. PubMed ID: 15054101

Craigen, W. J. and Graham, B. H. (2008). Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions. J Bioenerg Biomembr 40(3): 207-212. PubMed ID: 18622693

De Pinto, V., Benz, R., Caggese, C. and Palmieri, F. (1989). Characterization of the mitochondrial porin from Drosophila melanogaster. Biochim Biophys Acta 987(1): 1-7. PubMed ID: 2480813

Graham, B. H., Li, Z., Alesii, E. P., Versteken, P., Lee, C., Wang, J. and Craigen, W. J. (2010). Neurologic dysfunction and male infertility in Drosophila porin mutants: a new model for mitochondrial dysfunction and disease. J Biol Chem 285(15): 11143-11153. PubMed ID: 20110367

Groebe, K., Klemm-Manns, M., Schwall, G. P., Hubenthal, H., Unterluggauer, H., Jansen-Durr, P., Tanguay, R. M., Morrow, G. and Schrattenholz, A. (2010). Age-dependent posttranslational modifications of voltage-dependent anion channel 1. Exp Gerontol 45(7-8): 632-637. PubMed ID: 20189493

Oliva, M., Messina, A., Ragone, G., Caggese, C. and De Pinto, V. (1998). Sequence and expression pattern of the Drosophila melanogaster mitochondrial porin gene: evidence of a conserved protein domain between fly and mouse. FEBS Lett 430(3): 327-332. PubMed ID: 9688565

Lee, S., Leung, H. T., Kim, E., Jang, J., Lee, E., Baek, K., Pak, W. L. and Yoon, J. (2007). Effects of a mutation in the Drosophila porin gene encoding mitochondrial voltage-dependent anion channel protein on phototransduction. Dev Neurobiol 67(11): 1533-1545. PubMed ID: 17525991

Leggio, L., Guarino, F., Magri, A., Accardi-Gheit, R., Reina, S., Specchia, V., Damiano, F., Tomasello, M. F., Tommasino, M. and Messina, A. (2018). Mechanism of translation control of the alternative Drosophila melanogaster Voltage Dependent Anion-selective Channel 1 mRNAs. Sci Rep 8(1): 5347. PubMed ID: 29593233

M'Angale, P. G. and Staveley, B. E. (2016). Loss of porin function in dopaminergic neurons of Drosophila is suppressed by Buffy. J Biomed Sci 23(1): 84. PubMed ID: 27881168

Messina, A., Neri, M., Perosa, F., Caggese, C., Marino, M., Caizzi, R. and De Pinto, V. (1996). Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster. FEBS Lett 384(1): 9-13. PubMed ID: 8797793

Park, J., Kim, Y., Choi, S., Koh, H., Lee, S. H., Kim, J. M. and Chung, J. (2010). Drosophila Porin/VDAC affects mitochondrial morphology. PLoS One 5(10): e13151. PubMed ID: 20949033

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

date revised: 11 November 2018

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