ESCRT-dependent protein sorting is required for the viability of yeast clathrin-mediated endocytosis mutants.

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin-mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin-associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2.

A Flow Cytometry-Based Phenotypic Screen To Identify Novel Endocytic Factors in .

Endocytosis is a fundamental process for internalizing material from the plasma membrane, including many transmembrane proteins that are selectively internalized depending on environmental conditions. In most cells, the main route of entry is clathrin-mediated endocytosis (CME), a process that involves the coordinated activity of over 60 proteins; however, there are likely as-yet unidentified proteins involved in cargo selection and/or regulation of endocytosis. We performed a mutagenic screen to identify novel endocytic genes in expressing the methionine permease Mup1 tagged with pHluorin (pHl), a pH-sensitive GFP variant whose fluorescence is quenched upon delivery to the acidic vacuole lumen.

Syp1 regulates the clathrin-mediated and clathrin-independent endocytosis of multiple cargo proteins through a novel sorting motif.

Internalization of proteins from the plasma membrane (PM) allows for cell-surface composition regulation, signaling of network modulation, and nutrient uptake. Clathrin-mediated endocytosis (CME) is a major internalization route for PM proteins. During CME, endocytic adaptor proteins bind cargoes at the cell surface and link them to the PM and clathrin coat.

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments.

DePFth Perception in Clathrin-Mediated Endocytosis.

The earliest stages of clathrin-coated structure (CCS) assembly involve the recruitment and stabilization of clathrin-binding adaptor proteins and the clathrin coat. In this issue of Developmental Cell, Ma et al. (2016) now identify transient protein interactions that form the basis of AP-2 adaptor complex stabilization, key to initiating CCS formation.

Vestiges of Ent3p/Ent5p function in the giardial epsin homolog.

An accurate way to characterize the functional potential of a protein is to analyze recognized protein domains encoded by the genes in a given group. The epsin N-terminal homology (ENTH) domain is an evolutionarily conserved protein module found primarily in proteins that participate in clathrin-mediated trafficking. In this work, we investigate the function of the single ENTH-containing protein from the protist Giardia lamblia by testing its function in Saccharomyces cerevisiae.

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.

α-Arrestins participate in cargo selection for both clathrin-independent and clathrin-mediated endocytosis.

Clathrin-mediated endocytosis (CME) is a well-studied mechanism to internalize plasma membrane proteins; however, to endocytose such cargo, most eukaryotic cells also use alternative clathrin-independent endocytic (CIE) pathways, which are less well characterized. The budding yeast Saccharomyces cerevisiae, a widely used model for studying CME, was recently shown to have a CIE pathway that requires the GTPase Rho1, the formin Bni1, and their regulators. Nevertheless, in both yeast and mammalian cells, the mechanisms underlying cargo selection in CME and CIE are only beginning to be understood.

Actin and endocytosis in budding yeast.

Endocytosis, the process whereby the plasma membrane invaginates to form vesicles, is essential for bringing many substances into the cell and for membrane turnover. The mechanism driving clathrin-mediated endocytosis (CME) involves > 50 different protein components assembling at a single location on the plasma membrane in a temporally ordered and hierarchal pathway. These proteins perform precisely choreographed steps that promote receptor recognition and clustering, membrane remodeling, and force-generating actin-filament assembly and turnover to drive membrane invagination and vesicle scission.

Pan1 regulates transitions between stages of clathrin-mediated endocytosis.

Endocytosis is a well-conserved process by which cells invaginate small portions of the plasma membrane to create vesicles containing extracellular and transmembrane cargo proteins. Dozens of proteins and hundreds of specific binding interactions are needed to coordinate and regulate these events. Saccharomyces cerevisiae is a powerful model system with which to study clathrin-mediated endocytosis (CME).

Epsin deficiency impairs endocytosis by stalling the actin-dependent invagination of endocytic clathrin-coated pits.

Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect.

Specific α-arrestins negatively regulate Saccharomyces cerevisiae pheromone response by down-modulating the G-protein-coupled receptor Ste2.

G-protein-coupled receptors (GPCRs) are integral membrane proteins that initiate responses to extracellular stimuli by mediating ligand-dependent activation of cognate heterotrimeric G proteins. In yeast, occupancy of GPCR Ste2 by peptide pheromone α-factor initiates signaling by releasing a stimulatory Gβγ complex (Ste4-Ste18) from its inhibitory Gα subunit (Gpa1). Prolonged pathway stimulation is detrimental, and feedback mechanisms have evolved that act at the receptor level to limit the duration of signaling and stimulate recovery from pheromone-induced G1 arrest, including upregulation of the expression of an α-factor-degrading protease (Bar1), a regulator of G-protein signaling protein (Sst2) that stimulates Gpa1-GTP hydrolysis, and Gpa1 itself.

Glucose starvation inhibits autophagy via vacuolar hydrolysis and induces plasma membrane internalization by down-regulating recycling.

Cellular energy influences all aspects of cellular function. Although cells can adapt to a gradual reduction in energy, acute energy depletion poses a unique challenge. Because acute depletion hampers the transport of new energy sources into the cell, the cell must use endogenous substrates to replenish energy after acute depletion.

Targeted disruption of an EH-domain protein endocytic complex, Pan1-End3.

Pan1 is a multi-domain scaffold that enables dynamic interactions with both structural and regulatory components of the endocytic pathway. Pan1 is composed of Eps15 Homology (EH) domains which interact with adaptor proteins, a central region that is responsible for its oligomerization and C-terminal binding sites for Arp2/3, F-actin, and type-I myosin motors. In this study, we have characterized the binding sites between Pan1 and its constitutive binding partner End3, another EH domain containing endocytic protein.

Pan1 is an intrinsically disordered protein with homotypic interactions.

The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction.

Conserved roles for yeast Rho1 and mammalian RhoA GTPases in clathrin-independent endocytosis.

Eukaryotic cells use numerous endocytic pathways for nutrient uptake, protein turnover and response to the extracellular environment. While clathrin-mediated endocytosis (CME) has been extensively studied in yeast and mammalian cells, recent studies in higher eukaryotes have described multiple clathrin-independent endocytic pathways that depend upon Rho family GTPases and their effector proteins. In contrast, yeast cells have been thought to rely solely on CME.

Distinct and separable activities of the endocytic clathrin-coat components Fcho1/2 and AP-2 in developmental patterning.

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show that the μ-homology domain of FCHO1/2 represents an endocytic interaction hub.

Existence of a novel clathrin-independent endocytic pathway in yeast that depends on Rho1 and formin.

Yeast is a powerful model organism for dissecting the temporal stages and choreography of the complex protein machinery during endocytosis. The only known mechanism for endocytosis in yeast is clathrin-mediated endocytosis, even though clathrin-independent endocytic pathways have been described in other eukaryotes. Here, we provide evidence for a clathrin-independent endocytic pathway in yeast.

Endocytic adaptors--social networking at the plasma membrane.

Receptor-mediated endocytosis is a dynamic process that is crucial for maintaining plasma membrane composition and controlling cell-signaling pathways. A variety of entry routes have evolved to ensure that the vast array of molecules on the cell surface can be differentially internalized by endocytosis. This diversity has extended to include a growing list of endocytic adaptor proteins, which are thought to initiate the internalization process.

Quantitative analysis of endocytosis with cytoplasmic pHluorin chimeras.

The pH-sensitive green fluorescent protein (GFP) variant pHluorin is typically fused to the extracellular domain of transmembrane proteins to monitor endocytosis. Here, we have turned pHluorin inside-out, and show that cytoplasmic fusions of pHluorin are effective quantitative reporters for endocytosis and multivesicular body (MVB) sorting. In yeast in particular, fusion of GFP and its variants on the extracellular side of transmembrane proteins can result in perturbed trafficking.

A novel, retromer-independent role for sorting nexins 1 and 2 in RhoG-dependent membrane remodeling.

The sorting nexins SNX1 and SNX2 are members of the retromer complex involved in protein sorting within the endocytic pathway. While retromer-dependent functions of SNX1 and SNX2 have been well documented, potential retromer-independent roles remain unclear. Here, we show that SNX1 and SNX2 interact with the Rac1 and RhoG guanine nucleotide exchange factor Kalirin-7.

The F-BAR protein Syp1 negatively regulates WASp-Arp2/3 complex activity during endocytic patch formation.

Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators.

The function of yeast epsin and Ede1 ubiquitin-binding domains during receptor internalization.

The formation of a primary endocytic vesicle is a dynamic process involving the transient organization of adaptor and scaffold proteins at the plasma membrane. Epsins and Eps15-like proteins are ubiquitin-binding proteins that act early in this process. The yeast epsins, Ent1 and Ent2, carry functional ubiquitin-interacting motifs (UIMs), whereas the yeast Eps15-like protein, Ede1, has a C-terminal ubiquitin-associated (UBA) domain.

Syp1 is a conserved endocytic adaptor that contains domains involved in cargo selection and membrane tubulation.

Internalization of diverse transmembrane cargos from the plasma membrane requires a similarly diverse array of specialized adaptors, yet only a few adaptors have been characterized. We report the identification of the muniscin family of endocytic adaptors that is conserved from yeast to human beings. Solving the structures of yeast muniscin domains confirmed the unique combination of an N-terminal domain homologous to the crescent-shaped membrane-tubulating EFC/F-BAR domains and a C-terminal domain homologous to cargo-binding mu homology domains (muHDs).