The MyLO CRISPR-Cas9 toolkit: a markerless yeast localization and overexpression CRISPR-Cas9 toolkit.

The genetic tractability of the yeast Saccharomyces cerevisiae has made it a key model organism for basic research and a target for metabolic engineering. To streamline the introduction of tagged genes and compartmental markers with powerful Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - CRISPR-associated protein 9 (Cas9)-based genome editing tools, we constructed a Markerless Yeast Localization and Overexpression (MyLO) CRISPR-Cas9 toolkit with 3 components: (1) a set of optimized Streptococcus pyogenes Cas9-guide RNA expression vectors with 5 selectable markers and the option to either preclone or cotransform the gRNAs; (2) vectors for the one-step construction of integration cassettes expressing an untagged or green fluorescent protein/red fluorescent protein/hemagglutinin-tagged gene of interest at one of 3 levels, supporting localization and overexpression studies; and (3) integration cassettes containing moderately expressed green fluorescent protein- or red fluorescent protein-tagged compartmental markers for colocalization experiments. These components allow rapid, high-efficiency genomic integrations and modifications with only transient selection for the Cas9 vector, resulting in markerless transformations. To demonstrate the ease of use, we applied our complete set of compartmental markers to colabel all target subcellular compartments with green fluorescent protein and red fluorescent protein. Thus, the MyLO toolkit packages CRISPR-Cas9 technology into a flexible, optimized bundle that allows the stable genomic integration of DNA with the ease of use approaching that of transforming plasmids.

Functional expression of opioid receptors and other human GPCRs in yeast engineered to produce human sterols.

The yeast Saccharomyces cerevisiae is powerful for studying human G protein-coupled receptors as they can be coupled to its mating pathway. However, some receptors, including the mu opioid receptor, are non-functional, which may be due to the presence of the fungal sterol ergosterol instead of cholesterol. Here we engineer yeast to produce cholesterol and introduce diverse mu, delta, and kappa opioid receptors to create sensitive opioid biosensors that recapitulate agonist binding profiles and antagonist inhibition. Additionally, human mu opioid receptor variants, including those with clinical relevance, largely display expected phenotypes. By testing mu opioid receptor-based biosensors with systematically adjusted cholesterol biosynthetic intermediates, we relate sterol profiles to biosensor sensitivity. Finally, we apply sterol-modified backgrounds to other human receptors revealing sterol influence in SSTR5, 5-HTR4, FPR1, and NPY1R signaling. This work provides a platform for generating human G protein-coupled receptor-based biosensors, facilitating receptor deorphanization and high-throughput screening of receptors and effectors.

A VPS13D spastic ataxia mutation disrupts the conserved adaptor-binding site in yeast Vps13.

Mutations in each of the four human VPS13 (VPS13A-D) proteins are associated with distinct neurological disorders: chorea-acanthocytosis, Cohen syndrome, early-onset Parkinson's disease and spastic ataxia. Recent evidence suggests that the different VPS13 paralogs transport lipids between organelles at different membrane contact sites. How each VPS13 isoform is targeted to organelles is not known. We have shown that the localization of yeast Vps13 protein to membranes requires a conserved six-repeat region, the Vps13 Adaptor Binding (VAB) domain, which binds to organelle-specific adaptors. Here, we use a systematic mutagenesis strategy to determine the role of each repeat in recognizing each known adaptor. Our results show that mutation of invariant asparagines in repeats 1 and 6 strongly impacts the binding of all adaptors and blocks Vps13 membrane recruitment. However, we find that repeats 5-6 are sufficient for localization and interaction with adaptors. This supports a model where a single adaptor-binding site is found in the last two repeats of the VAB domain, while VAB domain repeat 1 may influence domain conformation. Importantly, a disease-causing mutation in VPS13D, which maps to the highly conserved asparagine residue in repeat 6, blocks adaptor binding and Vps13 membrane recruitment when modeled in yeast. Our findings are consistent with a conserved adaptor binding role for the VAB domain and suggest the presence of as-yet-unidentified adaptors in both yeast and humans.

Competitive organelle-specific adaptors recruit Vps13 to membrane contact sites.

The regulated expansion of membrane contact sites, which mediate the nonvesicular exchange of lipids between organelles, requires the recruitment of additional contact site proteins. Yeast Vps13 dynamically localizes to membrane contacts that connect the ER, mitochondria, endosomes, and vacuoles and is recruited to the prospore membrane in meiosis, but its targeting mechanism is unclear. In this study, we identify the sorting nexin Ypt35 as a novel adaptor that recruits Vps13 to endosomal and vacuolar membranes. We characterize an interaction motif in the Ypt35 N terminus and identify related motifs in the prospore membrane adaptor Spo71 and the mitochondrial membrane protein Mcp1. We find that Mcp1 is a mitochondrial adaptor for Vps13, and the Vps13-Mcp1 interaction, but not Ypt35, is required when ER-mitochondria contacts are lost. All three adaptors compete for binding to a conserved six-repeat region of Vps13 implicated in human disease. Our results support a competition-based model for regulating Vps13 localization at cellular membranes.

The chaperone Chs7 forms a stable complex with Chs3 and promotes its activity at the cell surface.

The polytopic yeast protein Chs3 (chitin synthase III) relies on a dedicated membrane-localized chaperone, Chs7, for its folding and expression at the cell surface. In the absence of Chs7, Chs3 forms high molecular weight aggregates and is retained in the endoplasmic reticulum (ER). Chs7 was reported to be an ER resident protein, but its role in Chs3 folding and transport was not well characterized. Here, we show that Chs7 itself exits the ER and localizes with Chs3 at the bud neck and intracellular compartments. We identified mutations in the Chs7 C-terminal cytosolic domain that do not affect its chaperone function, but cause it to dissociate from Chs3 at a post-ER transport step. Mutations that prevent the continued association of Chs7 with Chs3 do not block delivery of Chs3 to the cell surface, but dramatically reduce its catalytic activity. This suggests that Chs7 engages in functionally distinct interactions with Chs3 to first promote its folding and ER exit, and subsequently to regulate its activity at the plasma membrane.

Quantitative high-content imaging identifies novel regulators of Neo1 trafficking at endosomes.

P4-ATPases are a family of putative phospholipid flippases that regulate lipid membrane asymmetry, which is important for vesicle formation. Two yeast flippases, Drs2 and Neo1, have nonredundant functions in the recycling of the synaptobrevin-like v-SNARE Snc1 from early endosomes. Drs2 activity is needed to form vesicles and regulate its own trafficking, suggesting that flippase activity and localization are linked. However, the role of Neo1 in endosomal recycling is not well characterized. To identify novel regulators of Neo1 trafficking and activity at endosomes, we first identified mutants with impaired recycling of a Snc1-based reporter and subsequently used high-content microscopy to classify these mutants based on the localization of Neo1 or its binding partners, Mon2 and Dop1. This analysis identified a role for Arl1 in stabilizing the Mon2/Dop1 complex and uncovered a new function for Vps13 in early endosome recycling and Neo1 localization. We further showed that the cargo-selective sorting nexin Snx3 is required for Neo1 trafficking and identified an Snx3 sorting motif in the Neo1 N-terminus. Of importance, the Snx3-dependent sorting of Neo1 was required for the correct sorting of another Snx3 cargo protein, suggesting that the incorporation of Neo1 into recycling tubules may influence their formation.

Cargo selectivity of yeast sorting nexins.

Sorting nexins are PX domain-containing proteins that bind phospholipids and often act in membrane trafficking where they help to select cargo. However, the functions and cargo specificities of many sorting nexins are unknown. Here, a high-throughput imaging screen was used to identify new sorting nexin cargo in the yeast Saccharomyces cerevisiae. Deletions of 9 different sorting nexins were screened for mislocalization of a set of green fluorescent protein (GFP)-tagged membrane proteins found at the plasma membrane, Golgi or endosomes. This identified 27 proteins that require 1 or more sorting nexins for their correct localization, 23 of which represent novel sorting nexin cargo. Nine hits whose sorting was dependent on Snx4, the sorting nexin-containing retromer complex, or both retromer and Snx3, were examined in detail to search for potential sorting motifs. We identified cytosolic domains of Ear1, Ymd8 and Ymr010w that conferred retromer-dependent sorting on a chimeric reporter and identified conserved residues required for this sorting in a functional assay. This work defined a consensus sequence for retromer and Snx3-dependent sorting.

The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting.

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type-specific expression of alternate µ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the µ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the µ chain. Here we show that the variant µ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes.

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain-containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.