Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and co-localization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations.

Average collision velocity of single yeast cells during electrochemically induced impacts.

We recorded current-time (-) profiles for oxidizing ferrocyanide (FCN) while spherical yeast cells of radius ( ≈ 2 μm) collided with disk ultramicroelectrodes (UMEs) of increasing radius ( ≈ 12-45 μm). Collision signals appear as minority steps and majority blips of decreased current overlayed on the - baseline when cells block ferrocyanide flux (). We assigned steps to adsorption events and blips to bouncing collisions or contactless passages.

A CIE change in our understanding of endocytic mechanisms.

The past six decades have seen major advances in our understanding of endocytosis, ranging from descriptive studies based on electron microscopy to biochemical and genetic characterization of factors required for vesicle formation. Most studies focus on clathrin as the major coat protein; indeed, clathrin-mediated endocytosis (CME) is the primary pathway for internalization. Clathrin-independent (CIE) pathways also exist, although mechanistic understanding of these pathways remains comparatively elusive.

Actin- and microtubule-based motors contribute to clathrin-independent endocytosis in yeast.

Most eukaryotic cells utilize clathrin-mediated endocytosis as well as multiple clathrin-independent pathways to internalize proteins and membranes. Although clathrin-mediated endocytosis has been studied extensively and many machinery proteins have been identified, clathrin-independent pathways remain poorly characterized by comparison. We previously identified the first known yeast clathrin-independent endocytic pathway, which relies on the actin-modulating GTPase Rho1, the formin Bni1 and unbranched actin filaments, but does not require the clathrin coat or core clathrin machinery proteins.

Yeast Models of Amyotrophic Lateral Sclerosis Type 8 Mimic Phenotypes Seen in Mammalian Cells Expressing Mutant VAPB.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disease that results in the loss of motor neurons and can occur sporadically or due to genetic mutations. Among the 30 genes linked to familial ALS, a P56S mutation in , an ER-resident protein that functions at membrane contact sites, causes ALS type 8. Mammalian cells expressing VAPB have distinctive phenotypes, including ER collapse, protein and/or membrane-containing inclusions, and sensitivity to ER stress.

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.

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.

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.

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.

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.

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.

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.

FFAT rescues VAPA-mediated inhibition of ER-to-Golgi transport and VAPB-mediated ER aggregation.

The VAMP-associated proteins termed VAP are a small gene family of proteins characterised by the presence of an N-terminal major sperm protein (MSP) domain. The P56S mutation of the B isoform (VAPB) has been linked to late-onset amyotrophic lateral sclerosis (ALS8) and its expression causes formation of large ER aggregates. Overexpression of the wild-type A isoform (VAPA) but not the B isoform (VAPB), inhibited ER-to-Golgi transport of membrane proteins.

Disruption of Golgi morphology and trafficking in cells expressing mutant prenylated rab acceptor-1.

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization.