Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. An important means of communication is through membrane contact sites, where two organelles come into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). We show that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication.

Peroxisomes are organelles with crucial functions in oxidative metabolism. To correctly target to peroxisomes, proteins require specialized targeting signals. A mystery in the field is the sorting of proteins that carry a targeting signal for peroxisomes and as well as for other organelles, such as mitochondria or the endoplasmic reticulum (ER). Exploring several of these proteins in fungal model systems, we observed that they can act as tethers bridging organelles together to create contact sites. We show that in Saccharomyces cerevisiae this mode of tethering involves the peroxisome import machinery, the ER-mitochondria encounter structure (ERMES) at mitochondria and the guided entry of tail-anchored proteins (GET) pathway at the ER. Our findings introduce a previously unexplored concept of how dual affinity proteins can regulate organelle attachment and communication.

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.

Mitochondrial functions are tightly regulated by nuclear activity, requiring extensive communication between these organelles. One way by which organelles can communicate is through contact sites, areas of close apposition held together by tethering molecules. While many contacts have been characterized in yeast, the contact between the nucleus and mitochondria was not previously identified. Using fluorescence and electron microscopy in S. cerevisiae, we demonstrate specific areas of contact between the two organelles. Using a high-throughput screen, we uncover a role for the uncharacterized protein Ybr063c, which we have named Cnm1 (contact nucleus mitochondria 1), as a molecular tether on the nuclear membrane. We show that Cnm1 mediates contact by interacting with Tom70 on mitochondria. Moreover, Cnm1 abundance is regulated by phosphatidylcholine, enabling the coupling of phospholipid homeostasis with contact extent. The discovery of a molecular mechanism that allows mitochondrial crosstalk with the nucleus sets the ground for better understanding of mitochondrial functions in health and disease.

Eukaryotic cells have evolved organelles that allow the compartmentalization and regulation of metabolic processes. Knowledge of molecular mechanisms that allow temporal and spatial organization of enzymes within organelles is therefore crucial for understanding eukaryotic metabolism. Here, we show that the yeast malate dehydrogenase 2 (Mdh2) is dually localized to the cytosol and to peroxisomes and is targeted to peroxisomes via association with Mdh3 and a Pex5-dependent piggybacking mechanism. This dual localization of Mdh2 contributes to our understanding of the glyoxylate cycle and provides a new perspective on compartmentalization of cellular metabolism, which is critical for the perception of metabolic disorders and aging.

Approximately half of eukaryotic proteins reside in organelles. To reach their correct destination, such proteins harbor targeting signals recognized by dedicated targeting pathways. It has been shown that differences in targeting signals alter the efficiency in which proteins are recognized and targeted. Since multiple proteins compete for any single pathway, such differences can affect the priority for which a protein is catered. However, to date the entire repertoire of proteins with targeting priority, and the mechanisms underlying it, have not been explored for any pathway. Here we developed a systematic tool to study targeting priority and used the Pex5-mediated targeting to yeast peroxisomes as a model. We titrated Pex5 out by expressing high levels of a Pex5-cargo protein and examined how the localization of each peroxisomal protein is affected. We found that while most known Pex5 cargo proteins were outcompeted, several cargo proteins were not affected, implying that they have high targeting priority. This priority group was dependent on metabolic conditions. We dissected the mechanism of priority for these proteins and suggest that targeting priority is governed by different parameters, including binding affinity of the targeting signal to the cargo factor, the number of binding interfaces to the cargo factor, and more. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.

Crucial metabolic functions of peroxisomes rely on a variety of peroxisomal membrane proteins (PMPs). While mRNA transcripts of PMPs were shown to be colocalized with peroxisomes, the process by which PMPs efficiently couple translation with targeting to the peroxisomal membrane remained elusive. Here, we combine quantitative electron microscopy with proximity-specific ribosome profiling and reveal that translation of specific PMPs occurs on the surface of peroxisomes in the yeast Saccharomyces cerevisiae. This places peroxisomes alongside chloroplasts, mitochondria, and the endoplasmic reticulum as organelles that use localized translation for ensuring correct insertion of hydrophobic proteins into their membranes. Moreover, the correct targeting of these transcripts to peroxisomes is crucial for peroxisomal and cellular function, emphasizing the importance of localized translation for cellular physiology.

The understanding that organelles are not floating in the cytosol, but rather held in an organized yet dynamic interplay through membrane contact sites, is altering the way we grasp cell biological phenomena. However, we still have not identified the entire repertoire of contact sites, their tethering molecules and functions. To systematically characterize contact sites and their tethering molecules here we employ a proximity detection method based on split fluorophores and discover four potential new yeast contact sites. We then focus on a little-studied yet highly disease-relevant contact, the Peroxisome-Mitochondria (PerMit) proximity, and uncover and characterize two tether proteins: Fzo1 and Pex34. We genetically expand the PerMit contact site and demonstrate a physiological function in β-oxidation of fatty acids. Our work showcases how systematic analysis of contact site machinery and functions can deepen our understanding of these structures in health and disease.

The unfolded protein response (UPR) allows cells to adjust secretory pathway capacity according to need. Ire1, the endoplasmic reticulum (ER) stress sensor and central activator of the UPR is conserved from the budding yeast Saccharomyces cerevisiae to humans. Under ER stress conditions, Ire1 clusters into foci that enable optimal UPR activation. To discover factors that affect Ire1 clustering, we performed a high-content screen using a whole-genome yeast mutant library expressing Ire1-mCherry. We imaged the strains following UPR induction and found 154 strains that displayed alterations in Ire1 clustering. The hits were enriched for iron and heme effectors and binding proteins. By performing pharmacological depletion and repletion, we confirmed that iron (Fe3+) affects UPR activation in both yeast and human cells. We suggest that Ire1 clustering propensity depends on membrane composition, which is governed by heme-dependent biosynthesis of sterols. Our findings highlight the diverse cellular functions that feed into the UPR and emphasize the cross-talk between organelles required to concertedly maintain homeostasis.

Loss of the endoplasmic reticulum (ER)-mitochondria encounter structure (ERMES) complex that resides in contact sites between the yeast ER and mitochondria leads to impaired respiration; however, the reason for that is not clear. We find that in ERMES null mutants, there is an increase in the level of mRNAs encoding for biosynthetic enzymes of coenzyme Q6 (CoQ6), an essential electron carrier of the mitochondrial respiratory chain. We show that the mega complexes involved in CoQ6 biosynthesis (CoQ synthomes) are destabilized in ERMES mutants. This, in turn, affects the level and distribution of CoQ6 within the cell, resulting in reduced mitochondrial CoQ6. We suggest that these outcomes contribute to the reduced respiration observed in ERMES mutants. Fluorescence microscopy experiments demonstrate close proximity between the CoQ synthome and ERMES, suggesting a spatial coordination. The involvement of the ER-mitochondria contact site in regulation of CoQ6 biogenesis highlights an additional level of communication between these two organelles.

Membrane contact sites are specialized platforms formed between most organelles that enable them to exchange metabolites and influence the dynamics of each other. The yeast vacuole is a degradative organelle equivalent to the lysosome in higher eukaryotes with important roles in ion homeostasis and metabolism. Using a high-content microscopy screen, we identified Ymr160w (Cvm1, for contact of the vacuole membrane 1) as a novel component of three different contact sites of the vacuole: with the nuclear endoplasmic reticulum, the mitochondria, and the peroxisomes. At the vacuole-mitochondria contact site, Cvm1 acts as a tether independently of previously known tethers. We show that changes in Cvm1 levels affect sphingolipid homeostasis, altering the levels of multiple sphingolipid classes and the response of sphingolipid-sensing signaling pathways. Furthermore, the contact sites formed by Cvm1 are induced upon a decrease in sphingolipid levels. Altogether, our work identifies a novel protein that forms multiple contact sites and supports a role of lysosomal contacts in sphingolipid homeostasis.

Peroxisomes are organelles with vital functions in metabolism and their dysfunction is associated with human diseases. To fulfill their multiple roles, peroxisomes import nuclear-encoded matrix proteins, most carrying a peroxisomal targeting signal (PTS) 1. The receptor Pex5p recruits PTS1-proteins for import into peroxisomes; whether and how this process is posttranslationally regulated is unknown. Here, we identify 22 phosphorylation sites of Pex5p. Yeast cells expressing phospho-mimicking Pex5p-S507/523D (Pex5p2D) show decreased import of GFP with a PTS1. We show that the binding affinity between a PTS1-protein and Pex5p2D is reduced. An in vivo analysis of the effect of the phospho-mimicking mutant on PTS1-proteins revealed that import of most, but not all, cargos is affected. The physiological effect of the phosphomimetic mutations correlates with the binding affinity of the corresponding extended PTS1-sequences. Thus, we report a novel Pex5p phosphorylation-dependent mechanism for regulating PTS1-protein import into peroxisomes. In a broader view, this suggests that posttranslational modifications can function in fine-tuning the peroxisomal protein composition and, thus, cellular metabolism.

Mitochondria are unique organelles harboring two distinct membranes, the mitochondrial inner and outer membrane (MIM and MOM, respectively). Mitochondria comprise only a subset of metabolic pathways for the synthesis of membrane lipids; therefore most lipid species and their precursors have to be imported from other cellular compartments. One such import process is mediated by the ER mitochondria encounter structure (ERMES) complex. Both mitochondrial membranes surround the hydrophilic intermembrane space (IMS). Therefore, additional systems are required that shuttle lipids between the MIM and MOM. Recently, we identified the IMS protein Mcp2 as a high-copy suppressor for cells that lack a functional ERMES complex. To understand better how mitochondria facilitate transport and biogenesis of lipids, we searched for genetic interactions of this suppressor. We found that MCP2 has a negative genetic interaction with the gene TGL2 encoding a neutral lipid hydrolase. We show that this lipase is located in the intermembrane space of the mitochondrion and is imported via the Mia40 disulfide relay system. Furthermore, we show a positive genetic interaction of double deletion of MCP2 and PSD1, the gene encoding the enzyme that synthesizes the major amount of cellular phosphatidylethanolamine. Finally, we demonstrate that the nucleotide-binding motifs of the predicted atypical kinase Mcp2 are required for its proper function. Taken together, our data suggest that Mcp2 is involved in mitochondrial lipid metabolism and an increase of this involvement by overexpression suppresses loss of ERMES.

The endoplasmic reticulum (ER) is the site of synthesis of secreted and membrane proteins. To exit the ER, proteins are packaged into COPII vesicles through direct interaction with the COPII coat or aided by specific cargo receptors. Despite the fundamental role of such cargo receptors in protein traffic, only a few have been identified; their cargo spectrum is unknown and the signals they recognize remain poorly understood. We present here an approach we term "PAIRS" (pairing analysis of cargo receptors), which combines systematic genetic manipulations of yeast with automated microscopy screening, to map the spectrum of cargo for a known receptor or to uncover a novel receptor for a particular cargo. Using PAIRS we followed the fate of ∼150 cargos on the background of mutations in nine putative cargo receptors and identified novel cargo for most of these receptors. Deletion of the Erv14 cargo receptor affected the widest range of cargo. Erv14 substrates have a wide array of functions and structures; however, they are all membrane-spanning proteins of the late secretory pathway or plasma membrane. Proteins residing in these organelles have longer transmembrane domains (TMDs). Detailed examination of one cargo supported the hypothesis that Erv14 dependency reflects the length rather than the sequence of the TMD. The PAIRS approach allowed us to uncover new cargo for known cargo receptors and to obtain an unbiased look at specificity in cargo selection. Obtaining the spectrum of cargo for a cargo receptor allows a novel perspective on its mode of action. The rules that appear to guide Erv14 substrate recognition suggest that sorting of membrane proteins at multiple points in the secretory pathway could depend on the physical properties of TMDs. Such a mechanism would allow diverse proteins to utilize a few receptors without the constraints of evolving location-specific sorting motifs.

Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating environments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called 'LoQAtE' (Localizaiton and Quantitation Atlas of the yeast proteomE), available online at http://www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localization shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H₂O₂ and nitrogen starvation) and two genetic mutations [in the chaperonin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of localization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspectives on the significant role of proteins while combating external and internal fluctuations.