Four mutants defective in endocytosis were isolated by screening a collection of temperature-sensitive yeast mutants. Three mutations define new END genes: end5-1, end6-1, and end7-1. The fourth mutation is in END4, a gene identified previously. The end5-1, end6-1, and end7-1 mutations do not affect vacuolar protein localization, indicating that the defect in each mutant is specific for internalization at the plasma membrane. Interestingly, localization of actin patches on the plasma membrane is affected in each of the mutants. end5-1, end6-1, and end7-1 are allelic to VRP1, RVS161, and ACTI, respectively. VRP1 and RVS161 are required for correct actin localization and ACTi encodes actin. To our surprise, the end6 -1 mutation fails to complement the actl-l mutation. Disruption of the RVS167 gene, which is homologous to END6/RVS161 and which is also required for correct actin localization, also blocks endocytosis. The end7-1 mutant allele has a glycine 48 to aspartic acid substitution in the DNase I-binding loop of actin. We propose that Vrplp, Rvs161p, and Rvs167p are components of a cytoskeletal structure that contains actin and fimbrin and that is required for formation of endocytic vesicles at the plasma membrane.

Endocytosis in yeast requires actin and clathrin. Live cell imaging has previously shown that massive actin polymerization occurs concomitant with a slow 200-nm inward movement of the endocytic coat (Kaksonen, M., Y. Sun, and D.G. Drubin. 2003. Cell. 115:475–487). However, the nature of the primary endocytic profile in yeast and how clathrin and actin cooperate to generate an endocytic vesicle is unknown. In this study, we analyze the distribution of nine different proteins involved in endocytic uptake along plasma membrane invaginations using immunoelectron microscopy. We find that the primary endocytic profiles are tubular invaginations of up to 50 nm in diameter and 180 nm in length, which accumulate the endocytic coat components at the tip. Interestingly, significant actin labeling is only observed on invaginations longer than 50 nm, suggesting that initial membrane bending occurs before initiation of the slow inward movement. We also find that in the longest profiles, actin and the myosin-I Myo5p form two distinct structures that might be implicated in vesicle fission.

Type I myosins are thought to drive actin-dependent membrane motility, but the direct demonstration in vivo of their involvement in specific cellular processes has been difficult. Deletion of the genes MYO3 and MYO5, which encode the yeast type I myosins, almost abolished growth. A double-deleted mutant complemented with a MYO5 temperature-sensitive allele (myo5-1) showed a strong defect in the internalization step of receptor-mediated endocytosis, whereas the secretory pathway remained apparently unaffected. Thus, myosin I activity is required for a budding event in endocytosis but not for several other aspects of membrane traffic.

Summary Lipid droplets (LDs) are dynamic organelles that collect, store, and supply lipids [1]. LDs have a central role in the exchange of lipids occurring between the cell and the environment, and provide cells with substrates for energy metabolism, membrane synthesis, and production of lipid-derived molecules such as lipoproteins or hormones. However, lipid-derived metabolites also cause progressive lipotoxicity [2]; accumulation of reactive oxygen species (ROS), endoplasmic reticulum stress, mitochondrial malfunctioning, and cell death [2]. Intracellular accumulation of LDs is a hallmark of prevalent human diseases including obesity, steatosis, diabetes, myopathies, and arteriosclerosis [3]. Indeed, non-alcoholic fatty liver disease is the most common cause of abnormal hepatic function among adults [4, 5]. Lipotoxicity gradually promotes cellular ballooning and disarray, megamitochondria, and accumulation of Mallory’s hyaline in hepatocytes and inflammation, fibrosis, and cirrhosis in the liver. Here, using confocal microscopy, serial-block-face scanning electron microscopy, and flow-cytometry we show that LD accumulation is heterogeneous within a cell population and follows a positive skewed distribution. Lipid availability and fluctuations in biochemical networks controlling lipolysis, fatty acid oxidation, and protein synthesis, contribute to cell-to-cell heterogeneity. Critically, this reversible variability generates a subpopulation of cells that effectively collect and store lipids. This high-lipid subpopulation accumulates more LDs, more ROS, and reduces the risk of lipotoxicity to the population without impairing overall lipid homeostasis, since high-lipid cells can supply stored lipids to the other cells. In conclusion, we demonstrate fat storage compartmentalization within a cell population and propose that this is a protective social organization to reduce lipotoxicity.

contributed equally to this workThe yeast type I myosins (MYO3 and MYO5) are involved in endocytosis and in the polarization of the actin cytoskeleton. The tail of these proteins contains a Tail Homology 2 (TH2) domain that constitutes a putative actin-binding site. Because of the important mechanistic implications of a second ATP-independent actin-binding site, we analyzed its functional relevance in vivo. Even though the myosin tail interacts with actin, and this interaction seems functionally important, deletion of a major portion of the TH2 domain did not abolish interaction. In contrast, we found that the SH3 domain of Myo5p signi®cantly contributes to this interaction, implicating other proteins. We found that Vrp1p, the yeast homolog of WIP [Wiskott±Aldrich syndrome protein (WASP)-interacting protein], seems necessary to sustain the Myo5p tail±F-actin interaction. Consistent with recent results implicating the yeast type I myosins in regulating actin polymerization in vivo, we demonstrate that the C-terminal domain of Myo5p is able to induce cytosol-dependent actin polymerization in vitro, and that this activity requires both an intact Myo5p SH3 domain and Vrp1p.

Fluorescence live-cell imaging has temporally resolved the conserved choreography of more than 30 proteins involved in clathrin and actinmediated endocytic budding from the plasma membrane. However, the resolution of these studies is insufficient to unveil how the endocytic machinery actually drives membrane deformation in vivo. In this study, we use quantitative immuno-EM to introduce the temporal dimension to the ultrastructural analysis of membrane budding and define changes in the topography of the lipid bilayer coupled to the dynamics of endocytic proteins with unprecedented spatiotemporal resolution. Using this approach, we frame the emergence of membrane curvature with respect to the recruitment of endocytic factors and show that constriction of the invaginations correlates with translocation of membrane-sculpting proteins. Furthermore, we show that initial bending of the plasma membrane is independent of actin and clathrin polymerization and precedes building of an actin cap branched by the Arp2/3 complex. Finally, our data indicate that constriction and additional elongation of the endocytic profiles require the mechanochemical activity of the myosins-I. Altogether, this work provides major insights into the molecular mechanisms driving membrane deformation in a cellular context.

y-Zein is a maize storage protein synthesized by endosperm cells and stored together with a-and P-zeins in specialized organelles called protein bodies. Previous studies have shown that in maize there is only one type of protein body and it is derived directly from the endoplasmic reticulum (ER). In this article, we describe the domains of y-zein involved in ER retention and the domains involved in protein body formation. To identify the signal responsible for y-zein retention in ER-derived protein bodies, DNAs encoding various deletion mutants of y-zein were constructed and introduced into Arabidopsis as a heterologous system. By using pulse-chase experiments and immunoelectron microscopy, we demonstrated that the deletion of a proline-rich domain at the N terminus of y-zein puts an end to its retention in the ER; this resulted in the secretion of the mutated protein. The amino acid sequence of y-zein necessary for ER retention is the repeat domain composed of eight units of the hexapeptide PPPVHL. In addition, we obsenred that only those y-zein mutants that contained both the proline-rich repeat domain and the C-terminal cysteine-rich domain were able to form ER-derived protein bodies. We suggest that the retention of y-zein in the ER could be a result of a protein-protein association or a transient interaction of the repeat domain with ER membranes.

Genetic analysis of endocytosis in yeast early pointed to the essential role of actin in the uptake step. Efforts to identify the machinery involved demonstrated the important contribution of Arp2/3 and the myosins-I. Analysis of the process using livecell fluorescence microscopy and electron microscopy have recently contributed to refine molecular models explaining clathrin and actin-dependent endocytic uptake. Increasing evidence now also indicates that actin plays important roles in post-internalization events along the endocytic pathway in yeast, including transport of vesicles, motility of endosomes and vacuole fusion. This review describes the present knowledge state on the roles of actin in endocytosis in yeast and points to similarities and differences with analogous processes in mammals.