The Fast and the Furious: Golgi Contact Sites.

Contact sites are areas of close apposition between two membranes that coordinate nonvesicular communication between organelles. Such interactions serve a wide range of cellular functions from regulating metabolic pathways to executing stress responses and coordinating organelle inheritance. The past decade has seen a dramatic increase in information on certain contact sites, mostly those involving the endoplasmic reticulum. However, despite its central role in the secretory pathway, the Golgi apparatus and its contact sites remain largely unexplored. In this review, we discuss the current knowledge of Golgi contact sites and share our thoughts as to why Golgi contact sites are understudied. We also highlight what exciting future directions may exist in this emerging field.

Miro2 tethers the ER to mitochondria to promote mitochondrial fusion in tobacco leaf epidermal cells.

Mitochondria are highly pleomorphic, undergoing rounds of fission and fusion. Mitochondria are essential for energy conversion, with fusion favouring higher energy demand. Unlike fission, the molecular components involved in mitochondrial fusion in plants are unknown. Here, we show a role for the GTPase Miro2 in mitochondria interaction with the ER and its impacts on mitochondria fusion and motility. Mutations in AtMiro2's GTPase domain indicate that the active variant results in larger, fewer mitochondria which are attached more readily to the ER when compared with the inactive variant. These results are contrary to those in metazoans where Miro predominantly controls mitochondrial motility, with additional GTPases affecting fusion. Synthetically controlling mitochondrial fusion rates could fundamentally change plant physiology by altering the energy status of the cell. Furthermore, altering tethering to the ER could have profound effects on subcellular communication through altering the exchange required for pathogen defence.

Promethin Is a Conserved Seipin Partner Protein.

Seipin (BSCL2/SPG17) is a key factor in lipid droplet (LD) biology, and its dysfunction results in severe pathologies, including the fat storage disease Berardinelli-Seip congenital lipodystrophy type 2, as well as several neurological seipinopathies. Despite its importance for human health, the molecular role of seipin is still enigmatic. Seipin is evolutionarily conserved from yeast to humans. In yeast, seipin was recently found to cooperate with the lipid droplet organization (LDO) proteins, Ldo16 and Ldo45, two structurally-related proteins involved in LD function and identity that display remote homology to the human protein promethin/TMEM159. In this study, we show that promethin is indeed an LD-associated protein that forms a complex with seipin, and its localization to the LD surface can be modulated by seipin expression levels. We thus identify promethin as a novel seipin partner protein.

Miro1 - the missing link to peroxisome motility.

Peroxisomes are ubiquitous, highly dynamic, multifunctional compartments in eukaryotic cells, which perform key roles in cellular lipid metabolism and redox balance. Like other membrane-bound organelles, peroxisomes must move in the cellular landscape to perform localized functions, interact with other organelles and to properly distribute during cell division. However, our current knowledge of peroxisome motility in mammalian cells is still very limited. Recently, three independent studies have identified Miro1 as a regulator of peroxisome motility in mammalian cells. In these studies, the authors show that Miro1 is targeted to peroxisomes in several cell lines, in a process that relies on its interaction with the peroxisomal chaperone Pex19. Interestingly, however, different conclusions are drawn about which Miro1 isoforms are targeted to peroxisomes, how it interacts with Pex19 and most importantly, the type of motility Miro1 is regulating.

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.

Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation.

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.

Peroxisome Motility Measurement and Quantification Assay.

Organelle movement, distribution and interaction contribute to the organisation of the eukaryotic cell. Peroxisomes are multifunctional organelles which contribute to cellular lipid metabolism and ROS homeostasis. They distribute uniformly in mammalian cells and move along microtubules via kinesin and dynein motors. Their metabolic cooperation with mitochondria and the endoplasmic reticulum (ER) is essential for the ß-oxidation of fatty acids and the synthesis of myelin lipids and polyunsaturated fatty acids. A key assay to assess peroxisome motility in mammalian cells is the expression of a fluorescent fusion protein with a peroxisomal targeting signal (e.g., GFP-PTS1), which targets the peroxisomal matrix and allows live-cell imaging of peroxisomes. Here, we first present a protocol for the transfection of cultured mammalian cells with the peroxisomal marker EGFP-SKL to observe peroxisomes in living cells. This approach has revealed different motile behaviour of peroxisomes and novel insight into peroxisomal membrane dynamics (Rapp et al., 1996; Wiemer et al., 1997; Schrader et al., 2000). We then present a protocol which combines the live-cell approach with peroxisome motility measurements and quantification of peroxisome dynamics in mammalian cells. More recently, we used this approach to demonstrate that peroxisome motility and displacement is increased when a molecular tether, which associates peroxisomes with the ER, is lost (Costello et al., 2017b). Silencing of the peroxisomal acyl-CoA binding domain protein ACBD5, which interacts with ER-localised VAPB, increased peroxisome movement in skin fibroblasts, indicating that membrane contact sites can modulate organelle distribution and motility. The protocols described can be adapted to other cell types and organelles to measure and quantify organelle movement under different experimental conditions.

Peroxisomal ACBD4 interacts with VAPB and promotes ER-peroxisome associations.

Cooperation between cellular organelles such as mitochondria, peroxisomes and the ER is essential for a variety of important and diverse metabolic processes. Effective communication and metabolite exchange requires physical linkages between the organelles, predominantly in the form of organelle contact sites. At such contact sites organelle membranes are brought into close proximity by the action of molecular tethers, which often consist of specific protein pairs anchored in the membrane of the opposing organelles. Currently numerous tethering components have been identified which link the ER with multiple other organelles but knowledge of the factors linking the ER with peroxisomes is limited. Peroxisome-ER interplay is important because it is required for the biosynthesis of unsaturated fatty acids, ether-phospholipids and sterols with defects in these functions leading to severe diseases. Here, we characterize acyl-CoA binding domain protein 4 (ACBD4) as a tail-anchored peroxisomal membrane protein which interacts with the ER protein, vesicle-associated membrane protein-associated protein-B (VAPB) to promote peroxisome-ER associations.

Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

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.

ACBD5 and VAPB mediate membrane associations between peroxisomes and the ER.

Peroxisomes (POs) and the endoplasmic reticulum (ER) cooperate in cellular lipid metabolism and form tight structural associations, which were first observed in ultrastructural studies decades ago. PO-ER associations have been suggested to impact on a diverse number of physiological processes, including lipid metabolism, phospholipid exchange, metabolite transport, signaling, and PO biogenesis. Despite their fundamental importance to cell metabolism, the mechanisms by which regions of the ER become tethered to POs are unknown, in particular in mammalian cells. Here, we identify the PO membrane protein acyl-coenzyme A-binding domain protein 5 (ACBD5) as a binding partner for the resident ER protein vesicle-associated membrane protein-associated protein B (VAPB). We show that ACBD5-VAPB interaction regulates PO-ER associations. Moreover, we demonstrate that loss of PO-ER association perturbs PO membrane expansion and increases PO movement. Our findings reveal the first molecular mechanism for establishing PO-ER associations in mammalian cells and report a new function for ACBD5 in PO-ER tethering.