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.

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.

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores. The central role of Pex14p in peroxisomal matrix protein import processes renders it an obvious target for regulatory mechanisms such as protein phosphorylation. To explore this possibility, we examined the state of Pex14p phosphorylation in Saccharomyces cerevisiae. Phos-tag-SDS-PAGE of Pex14p affinity-purified from solubilized membranes revealed Pex14p as multi-phosphorylated protein. Using mass spectrometry, we identified 16 phosphorylation sites, with phosphorylation hot spots located in the N- and C-terminal regions of Pex14p. Analysis of phosphomimicking and non-phosphorylatable variants of Pex14p revealed a decreased import of GFP carrying a peroxisomal targeting signal type 1, indicating a functional relevance of Pex14p phosphorylation in peroxisomal matrix protein import. We show that this effect can be ascribed to the phosphomimicking mutation at serine 266 of Pex14p (Pex14p-S266D). We further screened the subcellular distribution of 23 native GFP-tagged peroxisomal matrix proteins by high-content fluorescence microscopy. Only Cit2p, the peroxisomal isoform of citrate synthase, was affected in the Pex14p-S266D mutant, showing increased cytosolic localization. Cit2p is part of the glyoxylate cycle, which is required for the production of essential carbohydrates when yeast is grown on non-fermentable carbon sources. Pex14p-S266 phosphosite mutants showed reversed growth phenotypes in oleic acid and ethanol with acetyl-CoA formed in peroxisomes and the cytosol, respectively. Overexpression of Cit2p rescued the growth phenotype of yeast cells expressing Pex14p-S266D in oleic acid. Our data indicate that phosphorylation of Pex14p at S266 provides a mechanism for controlling the peroxisomal import of Cit2p, which helps S. cerevisiae cells to adjust their carbohydrate metabolism according to the nutritional conditions.

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.

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.

The yeast Saccharomyces cerevisiae is ideal for systematic studies relying on collections of modified strains (libraries). Despite the significance of yeast libraries and the immense variety of available tags and regulatory elements, only a few such libraries exist, as their construction is extremely expensive and laborious. To overcome these limitations, we developed a SWAp-Tag (SWAT) method that enables one parental library to be modified easily and efficiently to give rise to an endless variety of libraries of choice. To showcase the versatility of the SWAT approach, we constructed and investigated a library of ∼1,800 strains carrying SWAT-GFP modules at the amino termini of endomembrane proteins and then used it to create two new libraries (mCherry and seamless GFP). Our work demonstrates how the SWAT method allows fast and effortless creation of yeast libraries, opening the door to new ways of systematically studying cell biology.

Tail-anchored (TA) proteins are post-translationally inserted into membranes. The TRC40 pathway targets TA proteins to the endoplasmic reticulum via a receptor comprised of WRB and CAML. TRC40 pathway clients have been identified using in vitro assays, however, the relevance of the TRC40 pathway in vivo remains unknown. We followed the fate of TA proteins in two tissue-specific WRB knockout mouse models and found that their dependence on the TRC40 pathway in vitro did not predict their reaction to receptor depletion in vivo. The SNARE syntaxin 5 (Stx5) was extremely sensitive to disruption of the TRC40 pathway. Screening yeast TA proteins with mammalian homologues, we show that the particular sensitivity of Stx5 is conserved, possibly due to aggregation propensity of its cytoplasmic domain. We establish that Stx5 is an autophagy target that is inefficiently membrane-targeted by alternative pathways. Our results highlight an intimate relationship between the TRC40 pathway and cellular proteostasis.

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.

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.

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are membrane-conjugated cell-surface proteins with diverse structural, developmental, and signaling functions and clinical relevance. Typically, after biosynthesis and attachment to the preassembled GPI anchor, GPI-APs rapidly leave the endoplasmic reticulum (ER) and rely on post-ER quality control. Terminally misfolded GPI-APs end up inside the vacuole/lysosome for degradation, but their trafficking itinerary to this organelle and the processes linked to their uptake by the vacuole/lysosome remain uncharacterized. In a yeast mutant that is lacking Pep4, a key vacuolar protease, several misfolded model GPI-APs accumulated in the vacuolar membrane. In the same mutant, macroautophagy and the multi-vesicular body (MVB) pathway were intact, hinting at a hitherto-unknown trafficking pathway for the degradation of misfolded GPI-APs. To unravel it, we used a genome-wide screen coupled to high-throughput fluorescence microscopy and followed the fate of the misfolded GPI-AP: Gas1∗. We found that components of the early secretory and endocytic pathways are involved in its targeting to the vacuole and that vacuolar transporter chaperones (VTCs), with roles in microautophagy, negatively affect the vacuolar uptake of Gas1∗. In support, we demonstrate that Gas1∗ internalizes from vacuolar membranes into membrane-bound intravacuolar vesicles prior to degradation. Our data link post-ER degradation with microautophagy.

Seventy years following the discovery of peroxisomes, their complete proteome, the peroxi-ome, remains undefined. Uncovering the peroxi-ome is crucial for understanding peroxisomal activities and cellular metabolism. We used high-content microscopy to uncover peroxisomal proteins in the model eukaryote - Saccharomyces cerevisiae. This strategy enabled us to expand the known peroxi-ome by ~40% and paved the way for performing systematic, whole-organellar proteome assays. By characterizing the sub-organellar localization and protein targeting dependencies into the organelle, we unveiled non-canonical targeting routes. Metabolomic analysis of the peroxi-ome revealed the role of several newly identified resident enzymes. Importantly, we found a regulatory role of peroxisomes during gluconeogenesis, which is fundamental for understanding cellular metabolism. With the current recognition that peroxisomes play a crucial part in organismal physiology, our approach lays the foundation for deep characterization of peroxisome function in health and disease.

Planarians have long been studied for their regenerative abilities. Moving forward, tools for ectopic expression of non-native proteins will be of substantial value. Using a luminescent reporter to overcome the strong autofluorescence of planarian tissues, we demonstrate heterologous protein expression in planarian cells and live animals. Our approach is based on the introduction of mRNA through several nanotechnological and chemical transfection methods. We improve reporter expression by altering untranslated region (UTR) sequences and codon bias, facilitating the measurement of expression kinetics in both isolated cells and whole planarians using luminescence imaging. We also examine protein expression as a function of variations in the UTRs of delivered mRNA, demonstrating a framework to investigate gene regulation at the post-transcriptional level. Together, these advances expand the toolbox for the mechanistic analysis of planarian biology and establish a foundation for the development and expansion of transgenic techniques in this unique model system.