Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.

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.

O-mannosylation is implicated in protein quality control in Saccharomyces cerevisiae due to the attachment of mannose to serine and threonine residues of un- or misfolded proteins in the endoplasmic reticulum (ER). This process also designated as unfolded protein O-mannosylation (UPOM) that ends futile folding cycles and saves cellular resources is mainly mediated by protein O-mannosyltransferases Pmt1 and Pmt2. Here we describe a genetic screen for factors that influence O-mannosylation in yeast, using slow-folding green fluorescent protein (GFP) as a reporter. Our screening identifies the RNA binding protein brefeldin A resistance factor 1 (Bfr1) that has not been linked to O-mannosylation and ER protein quality control before. We find that Bfr1 affects O-mannosylation through changes in Pmt1 and Pmt2 protein abundance but has no effect on PMT1 and PMT2 transcript levels, mRNA localization to the ER membrane or protein stability. Ribosome profiling reveals that Bfr1 is a crucial factor for Pmt1 and Pmt2 translation thereby affecting unfolded protein O-mannosylation. Our results uncover a new level of regulation of protein quality control in the secretory pathway.

Identification of both stable and transient interactions is essential for understanding protein function and regulation. While assessing stable interactions is more straightforward, capturing transient ones is challenging. In recent years, sophisticated tools have emerged to improve transient interactor discovery, with many harnessing the power of evolved biotin ligases for proximity labelling. However, biotinylation-based methods have lagged behind in the model eukaryote, Saccharomyces cerevisiae, possibly due to the presence of several abundant, endogenously biotinylated proteins. In this study, we optimised robust biotin-ligation methodologies in yeast and increased their sensitivity by creating a bespoke technique for downregulating endogenous biotinylation, which we term ABOLISH (Auxin-induced BiOtin LIgase diminiSHing). We used the endoplasmic reticulum insertase complex (EMC) to demonstrate our approaches and uncover new substrates. To make these tools available for systematic probing of both stable and transient interactions, we generated five full-genome collections of strains in which every yeast protein is tagged with each of the tested biotinylation machineries, some on the background of the ABOLISH system. This comprehensive toolkit enables functional interactomics of the entire yeast proteome.

Sphingolipids (SL) and their metabolites play key roles both as structural components of membranes and as signaling molecules. Many of the key enzymes and regulators of SL metabolism were discovered using the yeast Saccharomyces cerevisiae, and based on the high degree of conservation, a number of mammalian homologs were identified. Although yeast continues to be an important tool for SL research, the complexity of SL structure and nomenclature often hampers the ability of new researchers to grasp the subtleties of yeast SL biology and discover new modulators of this intricate pathway. Moreover, the emergence of lipidomics by mass spectrometry has enabled the rapid identification of SL species in yeast and rendered the analysis of SL composition under various physiological and pathophysiological conditions readily amenable. However, the complex nomenclature of the identified species renders much of the data inaccessible to non-specialists. In this review, we focus on parsing both the classical SL nomenclature and the nomenclature normally used during mass spectrometry analysis, which should facilitate the understanding of yeast SL data and might shed light on biological processes in which SLs are involved. Finally, we discuss a number of putative roles of various yeast SL species.

Upon amino acid (AA) starvation and TOR inactivation, plasma-membrane-localized permeases rapidly undergo ubiquitination and internalization via the vacuolar protein sorting/multivesicular body (VPS-MVB) pathway and are degraded in the yeast vacuole. We now show that specific Golgi proteins are also directed to the vacuole under these conditions as part of a Golgi quality-control (GQC) process. The degradation of GQC substrates is dependent upon ubiquitination by the defective-for-SREBP-cleavage (DSC) complex, which was identified via genetic screening and includes the Tul1 E3 ligase. Using a model GQC substrate, GFP-tagged Yif1, we show that vacuolar targeting necessitates upregulation of the VPS pathway via proteasome-mediated degradation of the initial endosomal sorting complex required for transport, ESCRT-0, but not downstream ESCRT components. Thus, early cellular responses to starvation include the targeting of specific Golgi proteins for degradation, a phenomenon reminiscent of the inactivation of BTN1, the yeast Batten disease gene ortholog.

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.

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.

Recently, approaches have been developed for high-throughput identification of synthetic sick/lethal gene pairs. However, these are only a specific example of the broader phenomenon of epistasis, wherein the presence of one mutation modulates the phenotype of another. We present analysis techniques for generating high-confidence quantitative epistasis scores from measurements made using synthetic genetic array and epistatic miniarray profile (E-MAP) technology, as well as several tools for higher-level analysis of the resulting data that are greatly enhanced by the quantitative score and detection of alleviating interactions.

Pex3 has been proposed to be important for the exit of peroxisomal membrane proteins (PMPs) from the ER, based on the observation that PMPs accumulate at the ER in Saccharomyces cerevisiae pex3 mutant cells. Using a combination of microscopy and biochemical approaches, we show that a subset of the PMPs, including the receptor docking protein Pex14, localizes to membrane vesicles in S. cerevisiae pex3 cells. These vesicles are morphologically distinct from the ER and do not co-sediment with ER markers in cell fractionation experiments. At the vesicles, Pex14 assembles with other peroxins (Pex13, Pex17, and Pex5) to form a complex with a composition similar to the PTS1 import pore in wild-type cells. Fluorescence microscopy studies revealed that also the PTS2 receptor Pex7, the importomer organizing peroxin Pex8, the ubiquitin conjugating enzyme Pex4 with its recruiting PMP Pex22, as well as Pex15 and Pex25 co-localize with Pex14. Other peroxins (including the RING finger complex and Pex27) did not accumulate at these structures, of which Pex11 localized to mitochondria. In line with these observations, proteomic analysis showed that in addition to the docking proteins and Pex5, also Pex7, Pex4/Pex22 and Pex25 were present in Pex14 complexes isolated from pex3 cells. However, formation of the entire importomer was not observed, most likely because Pex8 and the RING proteins were absent in the Pex14 protein complexes. Our data suggest that peroxisomal membrane vesicles can form in the absence of Pex3 and that several PMPs can insert in these vesicles in a Pex3 independent manner.

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Proteomic and structural data as well as computational predictions indicate that up to 25% of yeast MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We experimentally characterized a set of 15 yeast MRPs in vivo and found that five use internal MTSs. Further analysis of a conserved model MRP, Mrp17/bS6m, revealed the identity of the internal targeting signal. Similar to conventional MTS-containing proteins, the internal sequence mediates binding to TOM complexes. The entire sequence of Mrp17 contains positive charges mediating translocation. The fact that these sequence properties could not be reliably predicted by standard methods shows that mitochondrial protein targeting is more versatile than expected. We hypothesize that structural constraints imposed by ribosome assembly interfaces may have disfavored N-terminal presequences and driven the evolution of internal targeting signals in MRPs.

Actively maintained close appositions between organelle membranes, also known as contact sites, enable the efficient transfer of biomolecules between cellular compartments. Several such sites have been described as well as their tethering machineries. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize contact site proteomes, we established a high-throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, several of which are poorly characterized, on the background of 1165 strains expressing a mCherry-tagged yeast protein that has a cellular punctate distribution (a hallmark of contact sites), under regulation of the strong TEF2 promoter. By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified three homologs of Vps13 and Atg2 that are residents of multiple contact sites. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell. Overall, our analysis expands the universe of contact site residents and effectors and creates a rich database to mine for new functions, tethers, and regulators.

Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in vivo), uses high-throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel-lctiv can provide direct information on substrate specificity even for highly homologous proteins.

Mitochondrial complex I (CI) is the largest multi-subunit oxidative phosphorylation (OXPHOS) protein complex. Recent availability of a high-resolution human CI structure, and from two non-human mammals, enabled predicting the impact of mutations on interactions involving each of the 44 CI subunits. However, experimentally assessing the impact of the predicted interactions requires an easy and high-throughput method. Here, we created such a platform by cloning all 37 nuclear DNA (nDNA) and 7 mitochondrial DNA (mtDNA)-encoded human CI subunits into yeast expression vectors to serve as both 'prey' and 'bait' in the split murine dihydrofolate reductase (mDHFR) protein complementation assay (PCA). We first demonstrated the capacity of this approach and then used it to examine reported pathological OXPHOS CI mutations that occur at subunit interaction interfaces. Our results indicate that a pathological frame-shift mutation in the MT-ND2 gene, causing the replacement of 126 C-terminal residues by a stretch of only 30 amino acids, resulted in loss of specificity in ND2-based interactions involving these residues. Hence, the split mDHFR PCA is a powerful assay for assessing the impact of disease-causing mutations on pairwise protein-protein interactions in the context of a large protein complex, thus offering a possible mechanistic explanation for the underlying pathogenicity.

LAG1 was the first longevity assurance gene discovered in Saccharomyces cerevisiae The Lag1 protein is a ceramide synthase and its homolog, Lac1, has a similar enzymatic function but no role in aging. Lag1 and Lac1 lie in an enzymatic branch point of the sphingolipid pathway that is interconnected by the activity of the C4 hydroxylase, Sur2. By uncoupling the enzymatic branch point and using lipidomic mass spectrometry, metabolic labeling and in vitro assays we show that Lag1 preferentially synthesizes phyto-sphingolipids. Using photo-bleaching experiments we show that Lag1 is uniquely required for the establishment of a lateral diffusion barrier in the nuclear envelope, which depends on phytoceramide. Given the role of this diffusion barrier in the retention of aging factors in the mother cell, we suggest that the different specificities of the two ceramide synthases, and the specific effect of Lag1 on asymmetrical inheritance, may explain why Δlag1 cells have an increased lifespan while Δlac1 cells do not.

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.

Yeast libraries revolutionized the systematic study of cell biology. To extensively increase the number of such libraries, we used our previously devised SWAp-Tag (SWAT) approach to construct a genome-wide library of ~5,500 strains carrying the SWAT NOP1promoter-GFP module at the N terminus of proteins. In addition, we created six diverse libraries that restored the native regulation, created an overexpression library with a Cherry tag, or enabled protein complementation assays from two fragments of an enzyme or fluorophore. We developed methods utilizing these SWAT collections to systematically characterize the yeast proteome for protein abundance, localization, topology, and interactions.

Cellular redox status affects diverse cellular functions, including proliferation, protein homeostasis, and aging. Thus, individual differences in redox status can give rise to distinct sub-populations even among cells with identical genetic backgrounds. Here, we have created a novel methodology to track redox status at single cell resolution using the redox-sensitive probe Grx1-roGFP2. Our method allows identification and sorting of sub-populations with different oxidation levels in either the cytosol, mitochondria or peroxisomes. Using this approach, we defined a redox-dependent heterogeneity of yeast cells and characterized growth, as well as proteomic and transcriptomic profiles of distinctive redox subpopulations. We report that, starting in late logarithmic growth, cells of the same age have a bi-modal distribution of oxidation status. A comparative proteomic analysis between these populations identified three key proteins, Hsp30, Dhh1, and Pnc1, which affect basal oxidation levels and may serve as first line of defense proteins in redox homeostasis.

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+) homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+) in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+) we could observe concurrent reduction in many Mn(2+)-related process in the ER lumen. Conversely, cytosolic Mn(2+)-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+) transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+)-dependent neurological disorders.

Translocation into the endoplasmic reticulum (ER) is an initial and crucial biogenesis step for all secreted and endomembrane proteins in eukaryotes. ER insertion can take place through the well-characterized signal recognition particle (SRP)-dependent pathway or the less-studied route of SRP-independent translocation. To better understand the prevalence of the SRP-independent pathway, we systematically defined the translocational dependence of the yeast secretome. By combining hydropathy-based analysis and microscopy, we uncovered that a previously unappreciated fraction of the yeast secretome translocates without the aid of the SRP. Furthermore, we validated a family of SRP-independent substrates-the glycosylphosphatidylinositol (GPI)-anchored proteins. Studying this family, we identified a determinant for ER targeting and uncovered a network of cytosolic proteins that facilitate SRP-independent targeting and translocation. These findings highlight the underappreciated complexity of SRP-independent translocation, which enables this pathway to efficiently cope with its extensive substrate flux.