Differential roles for ACBD4 and ACBD5 in peroxisome-ER interactions and lipid metabolism.

Peroxisomes and the endoplasmic reticulum (ER) are intimately linked subcellular organelles, physically connected at membrane contact sites. While collaborating in lipid metabolism, for example, of very long-chain fatty acids (VLCFAs) and plasmalogens, the ER also plays a role in peroxisome biogenesis. Recent work identified tethering complexes on the ER and peroxisome membranes that connect the organelles. These include membrane contacts formed via interactions between the ER protein VAPB (vesicle-associated membrane protein-associated protein B) and the peroxisomal proteins ACBD4 and ACBD5 (acyl-coenzyme A-binding domain protein). Loss of ACBD5 has been shown to cause a significant reduction in peroxisome-ER contacts and accumulation of VLCFAs. However, the role of ACBD4 and the relative contribution these two proteins make to contact site formation and recruitment of VLCFAs to peroxisomes remain unclear. Here, we address these questions using a combination of molecular cell biology, biochemical, and lipidomics analyses following loss of ACBD4 or ACBD5 in HEK293 cells. We show that the tethering function of ACBD5 is not absolutely required for efficient peroxisomal ß-oxidation of VLCFAs. We demonstrate that loss of ACBD4 does not reduce peroxisome-ER connections or result in the accumulation of VLCFAs. Instead, the loss of ACBD4 resulted in an increase in the rate of ß-oxidation of VLCFAs. Finally, we observe an interaction between ACBD5 and ACBD4, independent of VAPB binding. Overall, our findings suggest that ACBD5 may act as a primary tether and VLCFA recruitment factor, whereas ACBD4 may have regulatory functions in peroxisomal lipid metabolism at the peroxisome-ER interface.

PEX11ß and FIS1 cooperate in peroxisome division independently of mitochondrial fission factor.

Peroxisome membrane dynamics and division are essential to adapt the peroxisomal compartment to cellular needs. The peroxisomal membrane protein PEX11ß (also known as PEX11B) and the tail-anchored adaptor proteins FIS1 (mitochondrial fission protein 1) and MFF (mitochondrial fission factor), which recruit the fission GTPase DRP1 (dynamin-related protein 1, also known as DNML1) to both peroxisomes and mitochondria, are key factors of peroxisomal division. The current model suggests that MFF is essential for peroxisome division, whereas the role of FIS1 is unclear. Here, we reveal that PEX11ß can promote peroxisome division in the absence of MFF in a DRP1- and FIS1-dependent manner. We also demonstrate that MFF permits peroxisome division independently of PEX11ß and restores peroxisome morphology in PEX11ß-deficient patient cells. Moreover, targeting of PEX11ß to mitochondria induces mitochondrial division, indicating the potential for PEX11ß to modulate mitochondrial dynamics. Our findings suggest the existence of an alternative, MFF-independent pathway in peroxisome division and report a function for FIS1 in the division of peroxisomes. This article has an associated First Person interview with the first authors of the paper.

Controlling contacts-Molecular mechanisms to regulate organelle membrane tethering.

In recent years, membrane contact sites (MCS), which mediate interactions between virtually all subcellular organelles, have been extensively characterized and shown to be essential for intracellular communication. In this review essay, we focus on an emerging topic: the regulation of MCS. Focusing on the tether proteins themselves, we discuss some of the known mechanisms which can control organelle tethering events and identify apparent common regulatory hubs, such as the VAP interface at the endoplasmic reticulum (ER). We also highlight several currently hypothetical concepts, including the idea of tether oligomerization and redox regulation playing a role in MCS formation. We identify gaps in our current understanding, such as the identity of the majority of kinases/phosphatases involved in tether modification and conclude that a holistic approach-incorporating the formation of multiple MCS, regulated by interconnected regulatory modulators-may be required to fully appreciate the true complexity of these fascinating intracellular communication systems.

A role for Mitochondrial Rho GTPase 1 (MIRO1) in motility and membrane dynamics of peroxisomes.

Peroxisomes are dynamic organelles which fulfil essential roles in lipid and ROS metabolism. Peroxisome movement and positioning allows interaction with other organelles and is crucial for their cellular function. In mammalian cells, such movement is microtubule-dependent and mediated by kinesin and dynein motors. The mechanisms of motor recruitment to peroxisomes are largely unknown, as well as the role this plays in peroxisome membrane dynamics and proliferation. Here, using a combination of microscopy, live-cell imaging analysis and mathematical modelling, we identify a role for Mitochondrial Rho GTPase 1 (MIRO1) as an adaptor for microtubule-dependent peroxisome motility in mammalian cells. We show that MIRO1 is targeted to peroxisomes and alters their distribution and motility. Using a peroxisome-targeted MIRO1 fusion protein, we demonstrate that MIRO1-mediated pulling forces contribute to peroxisome membrane elongation and proliferation in cellular models of peroxisome disease. Our findings reveal a molecular mechanism for establishing peroxisome-motor protein associations in mammalian cells and provide new insights into peroxisome membrane dynamics in health and disease.

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells.

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.

Multi-localized Proteins: The Peroxisome-Mitochondria Connection.

Peroxisomes and mitochondria are dynamic, multifunctional organelles that play pivotal cooperative roles in the metabolism of cellular lipids and reactive oxygen species. Their functional interplay, the "peroxisome-mitochondria connection", also includes cooperation in anti-viral signalling and defence, as well as coordinated biogenesis by sharing key division proteins. In this review, we focus on multi-localised proteins which are shared by peroxisomes and mitochondria in mammals. We first outline the targeting and sharing of matrix proteins which are involved in metabolic cooperation. Next, we discuss shared components of peroxisomal and mitochondrial dynamics and division, and we present novel insights into the dual targeting of tail-anchored membrane proteins. Finally, we provide an overview of what is currently known about the role of shared membrane proteins in disease. What emerges is that sharing of proteins between these two organelles plays a key role in their cooperative functions which, based on new findings, may be more extensive than originally envisaged. Gaining a better insight into organelle interplay and the targeting of shared proteins is pivotal to understanding how organelle cooperation contributes to human health and disease.

eIF2ß is critical for eIF5-mediated GDP-dissociation inhibitor activity and translational control.

In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2ß that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2ß mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2ß mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2•GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2ß acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.

The yeast La related protein Slf1p is a key activator of translation during the oxidative stress response.

The mechanisms by which RNA-binding proteins control the translation of subsets of mRNAs are not yet clear. Slf1p and Sro9p are atypical-La motif containing proteins which are members of a superfamily of RNA-binding proteins conserved in eukaryotes. RIP-Seq analysis of these two yeast proteins identified overlapping and distinct sets of mRNA targets, including highly translated mRNAs such as those encoding ribosomal proteins. In paralell, transcriptome analysis of slf1Δ and sro9Δ mutant strains indicated altered gene expression in similar functional classes of mRNAs following loss of each factor. The loss of SLF1 had a greater impact on the transcriptome, and in particular, revealed changes in genes involved in the oxidative stress response. slf1Δ cells are more sensitive to oxidants and RIP-Seq analysis of oxidatively stressed cells enriched Slf1p targets encoding antioxidants and other proteins required for oxidant tolerance. To quantify these effects at the protein level, we used label-free mass spectrometry to compare the proteomes of wild-type and slf1Δ strains following oxidative stress. This analysis identified several proteins which are normally induced in response to hydrogen peroxide, but where this increase is attenuated in the slf1Δ mutant. Importantly, a significant number of the mRNAs encoding these targets were also identified as Slf1p-mRNA targets. We show that Slf1p remains associated with the few translating ribosomes following hydrogen peroxide stress and that Slf1p co-immunoprecipitates ribosomes and members of the eIF4E/eIF4G/Pab1p 'closed loop' complex suggesting that Slf1p interacts with actively translated mRNAs following stress. Finally, mutational analysis of SLF1 revealed a novel ribosome interacting domain in Slf1p, independent of its RNA binding La-motif. Together, our results indicate that Slf1p mediates a translational response to oxidative stress via mRNA-specific translational control.

The 4E-BP Caf20p Mediates Both eIF4E-Dependent and Independent Repression of Translation.

Translation initiation factor eIF4E mediates mRNA selection for protein synthesis via the mRNA 5'cap. A family of binding proteins, termed the 4E-BPs, interact with eIF4E to hinder ribosome recruitment. Mechanisms underlying mRNA specificity for 4E-BP control remain poorly understood. Saccharomyces cerevisiae 4E-BPs, Caf20p and Eap1p, each regulate an overlapping set of mRNAs. We undertook global approaches to identify protein and RNA partners of both 4E-BPs by immunoprecipitation of tagged proteins combined with mass spectrometry or next-generation sequencing. Unexpectedly, mass spectrometry indicated that the 4E-BPs associate with many ribosomal proteins. 80S ribosome and polysome association was independently confirmed and was not dependent upon interaction with eIF4E, as mutated forms of both Caf20p and Eap1p with disrupted eIF4E-binding motifs retain ribosome interaction. Whole-cell proteomics revealed Caf20p mutations cause both up and down-regulation of proteins and that many changes were independent of the 4E-binding motif. Investigations into Caf20p mRNA targets by immunoprecipitation followed by RNA sequencing revealed a strong association between Caf20p and mRNAs involved in transcription and cell cycle processes, consistent with observed cell cycle phenotypes of mutant strains. A core set of over 500 Caf20p-interacting mRNAs comprised of both eIF4E-dependent (75%) and eIF4E-independent targets (25%), which differ in sequence attributes. eIF4E-independent mRNAs share a 3' UTR motif. Caf20p binds all tested motif-containing 3' UTRs. Caf20p and the 3'UTR combine to influence ERS1 mRNA polysome association consistent with Caf20p contributing to translational control. Finally ERS1 3'UTR confers Caf20-dependent repression of expression to a heterologous reporter gene. Taken together, these data reveal conserved features of eIF4E-dependent Caf20p mRNA targets and uncover a novel eIF4E-independent mode of Caf20p binding to mRNAs that extends the regulatory role of Caf20p in the mRNA-specific repression of protein synthesis beyond its interaction with eIF4E.

Proliferation and fission of peroxisomes - An update.

In mammals, peroxisomes perform crucial functions in cellular metabolism, signalling and viral defense which are essential to the health and viability of the organism. In order to achieve this functional versatility peroxisomes dynamically respond to molecular cues triggered by changes in the cellular environment. Such changes elicit a corresponding response in peroxisomes, which manifests itself as a change in peroxisome number, altered enzyme levels and adaptations to the peroxisomal structure. In mammals the generation of new peroxisomes is a complex process which has clear analogies to mitochondria, with both sharing the same division machinery and undergoing a similar division process. How the regulation of this division process is integrated into the cell's response to different stimuli, the signalling pathways and factors involved, remains somewhat unclear. Here, we discuss the mechanism of peroxisomal fission, the contributions of the various division factors and examine the potential impact of post-translational modifications, such as phosphorylation, on the proliferation process. We also summarize the signalling process and highlight the most recent data linking signalling pathways with peroxisome proliferation.

Puf3p induces translational repression of genes linked to oxidative stress.

In response to stress, the translation of many mRNAs in yeast can change in a fashion discordant with the general repression of translation. Here, we use machine learning to mine the properties of these mRNAs to determine specific translation control signals. We find a strong association between transcripts acutely translationally repressed under oxidative stress and those associated with the RNA-binding protein Puf3p, a known regulator of cellular mRNAs encoding proteins targeted to mitochondria. Under oxidative stress, a PUF3 deleted strain exhibits more robust growth than wild-type cells and the shift in translation from polysomes to monosomes is attenuated, suggesting puf3Δ cells perceive less stress. In agreement, the ratio of reduced:oxidized glutathione, a major antioxidant and indicator of cellular redox state, is increased in unstressed puf3Δ cells but remains lower under stress. In untreated conditions, Puf3p migrates with polysomes rather than ribosome-free fractions, but this is lost under stress. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) of Puf3p targets following affinity purification shows Puf3p-mRNA associations are maintained or increased under oxidative stress. Collectively, these results point to Puf3p acting as a translational repressor in a manner exceeding the global translational response, possibly by temporarily limiting synthesis of new mitochondrial proteins as cells adapt to the stress.

Multiple Ways to Keep FFAT Under Control!

Peroxisomes and the ER are closely inter-connected organelles, which collaborate in the metabolism of lipids. In a recent research paper in the Journal of Cell Biology, we describe a novel mechanism by which peroxisome-ER membrane contact sites are regulated, via phosphorylation of the peroxisomal protein ACBD5. We found that the interaction between ACBD5 and the ER protein VAPB, which we have previously shown to form a tether complex at peroxisome-ER contacts, is controlled by phosphorylation of ACBD5 at two different sites of its FFAT motif - the VAPB binding site. We also identify the kinase GSK3-ß as being responsible for direct phosphorylation of ACBD5 to negatively regulate interaction with VAPB, leading to reduced peroxisome-ER contacts. In this article we provide additional insights into how this work, in combination with other studies on phosphorylation of VAP interactors, suggests a complex system of both positive and negative regulation of the FFAT motif via phosphorylation.

VAP Proteins - From Organelle Tethers to Pathogenic Host Interactors and Their Role in Neuronal Disease.

Vesicle-associated membrane protein (VAMP)-associated proteins (VAPs) are ubiquitous ER-resident tail-anchored membrane proteins in eukaryotic cells. Their N-terminal major sperm protein (MSP) domain faces the cytosol and allows them to interact with a wide variety of cellular proteins. Therefore, VAP proteins are vital to many cellular processes, including organelle membrane tethering, lipid transfer, autophagy, ion homeostasis and viral defence. Here, we provide a timely overview of the increasing number of VAPA/B binding partners and discuss the role of VAPA/B in maintaining organelle-ER interactions and cooperation. Furthermore, we address how viruses and intracellular bacteria hijack VAPs and their binding partners to induce interactions between the host ER and pathogen-containing compartments and support pathogen replication. Finally, we focus on the role of VAP in human disease and discuss how mutated VAPB leads to the disruption of cellular homeostasis and causes amyotrophic lateral sclerosis.

Regulating peroxisome-ER contacts via the ACBD5-VAPB tether by FFAT motif phosphorylation and GSK3ß.

Peroxisomes and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism. They form membrane contacts through interaction of the peroxisomal membrane protein ACBD5 (acyl-coenzyme A-binding domain protein 5) and the ER-resident protein VAPB (vesicle-associated membrane protein-associated protein B). ACBD5 binds to the major sperm protein domain of VAPB via its FFAT-like (two phenylalanines [FF] in an acidic tract) motif. However, molecular mechanisms, which regulate formation of these membrane contact sites, are unknown. Here, we reveal that peroxisome-ER associations via the ACBD5-VAPB tether are regulated by phosphorylation. We show that ACBD5-VAPB binding is phosphatase-sensitive and identify phosphorylation sites in the flanking regions and core of the FFAT-like motif, which alter interaction with VAPB-and thus peroxisome-ER contact sites-differently. Moreover, we demonstrate that GSK3ß (glycogen synthase kinase-3 ß) regulates this interaction. Our findings reveal for the first time a molecular mechanism for the regulation of peroxisome-ER contacts in mammalian cells and expand the current model of FFAT motifs and VAP interaction.

The diversity of ACBD proteins - From lipid binding to protein modulators and organelle tethers.

Members of the large multigene family of acyl-CoA binding domain containing proteins (ACBDs) share a conserved motif required for binding of Coenzyme A esterified fatty acids of various chain length. These proteins are present in the three kingdoms of life, and despite their predicted roles in cellular lipid metabolism, knowledge about the precise functions of many ACBD proteins remains scarce. Interestingly, several ACBD proteins are now suggested to function at organelle contact sites, and are recognized as host interaction proteins for different pathogens including viruses and bacteria. Here, we present a thorough phylogenetic analysis of the ACBD family and discuss their structure and evolution. We summarize recent findings on the various functions of animal and fungal ACBDs with particular focus on peroxisomes, the role of ACBD proteins at organelle membranes, and their increasing recognition as targets for pathogens.

Fluorescent tools to analyse peroxisome-ER interactions in mammalian cells.

Peroxisomes and the endoplasmic reticulum (ER) cooperate extensively in lipid-related metabolic pathways, and the ER also provides phospholipids to enable the peroxisomal membrane to expand prior to division. Recently, we identified peroxisomal proteins ACBD5 and ACBD4, and the ER protein VAPB as tethering components which physically interact to foster peroxisome-ER associations at membrane contact sites. Overexpression or loss of these tether proteins alters the extent of peroxisome-ER interactions, impacting on lipid exchange between these two compartments. To facilitate further studies into peroxisome-ER associations at the level of membrane contact sites, their role, composition and regulation, we have developed two fluorescence-based systems to monitor peroxisome-ER interactions. We modified a proximity ligation assay and a split-fluorescence reporter system using split superfolder green fluorescent protein. Using the proximity ligation assay we were able to measure changes in peroxisome-ER interactions whilst the split-fluorescence reporter was more limited and only allowed us to label ER-peroxisome contacts. We show that both techniques can be useful additions to the toolkit of methods to study peroxisome-ER associations and explore the relative merits of each.

Unloosing the Gordian knot of peroxisome formation.

Peroxisome biogenesis is governed by molecular machineries, which are either unique to peroxisomes or are partially shared with mitochondria. As peroxisomes have important protective functions in the cell, modulation of their number is important for human health and disease. Significant progress has been made towards our understanding of the mechanisms of peroxisome formation, revealing a remarkable plasticity of the peroxisome biogenesis pathway. Here we discuss most recent findings with particular focus on peroxisome formation in mammalian cells.

Intracellular redistribution of neuronal peroxisomes in response to ACBD5 expression.

Peroxisomes can be frequently found in proximity to other subcellular organelles such as the endoplasmic reticulum (ER), mitochondria or lysosomes. The tail-anchored protein ACBD5 was recently identified as part of a tethering complex at peroxisome-ER contact sites, interacting with the ER resident protein VAPB. Contact site disruption was found to significantly increase peroxisome motility, apparently interfering with intracellular positioning systems. Unlike other somatic cells, neurons have to distribute organelles across relatively long distances in order to maintain their extraordinary cellular polarity. Using confocal live imaging microscopy in cultured hippocampal neurons we observed that peroxisomes and mitochondria show a strikingly similar motility with approximately 10% performing microtubule-driven long range movements. In order to investigate if ER contacts influence overall peroxisome motility and cellular distribution patterns, hippocampal neurons were transfected with plasmids encoding ACBD5 to stimulate peroxisome-ER interactions. Overexpression of ACBD5 reduced peroxisomal long range movements in the neurites of the hippocampal cells by 70%, implying that ER attachment counteracts microtubule-driven peroxisome transport, while mitochondrial motility was unaffected. Moreover, the analyses of peroxisome distribution in fixed neurons unveiled a significant redistribution of peroxisomes towards the periphery of the perikaryon underneath the plasma membrane and into neurites, where peroxisomes are frequently found in close proximity to mitochondria. Surprisingly, further analysis of peroxisome and VAPB distribution upon ACBD5 expression did not reveal a substantial colocalization, implying this effect may be independent of VAPB. In line with these findings, expression of an ACBD5 variant unable to bind to VAPB still altered the localization of peroxisomes in the same way as the wild-type ACBD5. Thus, we conclude, that the VAPB-ACBD5 facilitated peroxisome-ER interaction is not responsible for the observed organelle redistribution in neurons. Rather, we suggest that additional ACBD5-binding proteins in neurons may tether peroxisomes to contact sites at or near the plasma membrane of neurons.

Archetypal transcriptional blocks underpin yeast gene regulation in response to changes in growth conditions.

The transcriptional responses of yeast cells to diverse stresses typically include gene activation and repression. Specific stress defense, citric acid cycle and oxidative phosphorylation genes are activated, whereas protein synthesis genes are coordinately repressed. This view was achieved from comparative transcriptomic experiments delineating sets of genes whose expression greatly changed with specific stresses. Less attention has been paid to the biological significance of 1) consistent, albeit modest, changes in RNA levels across multiple conditions, and 2) the global gene expression correlations observed when comparing numerous genome-wide studies. To address this, we performed a meta-analysis of 1379 microarray-based experiments in yeast, and identified 1388 blocks of RNAs whose expression changes correlate across multiple and diverse conditions. Many of these blocks represent sets of functionally-related RNAs that act in a coordinated fashion under normal and stress conditions, and map to global cell defense and growth responses. Subsequently, we used the blocks to analyze novel RNA-seq experiments, demonstrating their utility and confirming the conclusions drawn from the meta-analysis. Our results provide a new framework for understanding the biological significance of changes in gene expression: 'archetypal' transcriptional blocks that are regulated in a concerted fashion in response to external stimuli.