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. Many critical aspects of the CME mechanism are conserved from yeast to mammals and were first elucidated in yeast, demonstrating that it is a powerful system for studying endocytosis. In this review, we describe our current mechanistic understanding of each step in the process of yeast CME, and the essential roles played by actin polymerization at these sites, while providing a historical perspective of how the landscape has changed since the preceding version of the YeastBook was published 17 years ago (1997). Finally, we discuss the key unresolved issues and where future studies might be headed.
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. The key function of adaptors is to select the proteins that should be removed from the cell surface. Thus, they have a central role in defining the physiology of a cell. This has made the study of adaptor proteins a very active area of research that is ripe for exciting future discoveries. Here, we review recent work on how adaptors mediate endocytosis and address the following questions: what characteristics define an endocytic adaptor protein? What roles do these proteins fulfill in addition to selecting cargo and how might adaptors function in clathrin-independent endocytic pathways? Through the findings discussed in this Commentary, we hope to stimulate further characterization of known adaptors and expansion of the known repertoire by identification of new adaptors.
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). Pan1 is believed to be a scaffolding protein due to its interactions with numerous proteins that act throughout the endocytic process. Previous research characterized many Pan1 binding interactions, but due to Pan1's essential nature, the exact mechanisms of Pan1's function in endocytosis have been difficult to define. We created a novel Pan1-degron allele, Pan1-AID, in which Pan1 can be specifically and efficiently degraded in <1 h upon addition of the plant hormone auxin. The loss of Pan1 caused a delay in endocytic progression and weakened connections between the coat/actin machinery and the membrane, leading to arrest in CME. In addition, we determined a critical role for the central region of Pan1 in endocytosis and viability. The regions important for endocytosis and viability can be separated, suggesting that Pan1 may have a distinct role in the cell that is essential for viability.
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. In the yeast Saccharomyces cerevisiae, glucose starvation causes an acute depletion of intracellular energy that recovers during continued glucose starvation. However, how the cell replenishes energy during the early phase of glucose starvation is unknown. In this study, we investigated the role of pathways that deliver proteins and lipids to the vacuole during glucose starvation. We report that in response to glucose starvation, plasma membrane proteins are directed to the vacuole through reduced recycling at the endosomes. Furthermore, we found that vacuolar hydrolysis inhibits macroautophagy in a target of rapamycin complex 1-dependent manner. Accordingly, we found that endocytosis and hydrolysis are required for survival in glucose starvation, whereas macroautophagy is dispensable. Together, these results suggest that hydrolysis of components delivered to the vacuole independent of autophagy is the cell survival mechanism used by S. cerevisiae in response to glucose starvation.
Background: During energy starvation, cells utilize intracellular resources for survival; however, the resources used during glucose starvation are unknown.
Results: In glucose-starved yeast, vacuolar hydrolysis and endocytosis promote survival whereas autophagy is dispensable.
Conclusion: Vacuolar hydrolysis blocks autophagy and provides resources for survival during glucose starvation.
Significance: This new survival mechanism could protect cells from starvation in many situations.
Clathrin-dependent endocytosis allows cells to bring plasma membrane and extracellular molecules into the cell. Forming a clathrin-coated vesicle requires the sequential action of numerous factors, ...beginning with endocytic adaptors. Adaptors are thought to initiate the process in two ways: by selecting cargo for packaging into the vesicle and assembling the clathrin coat and other components necessary to shape the vesicle. Here, we review recent work focusing on the sequential and cooperative interactions of adaptors with their binding partners, and how adaptors contribute to initial stages of endocytic internalization. The regulation of adaptors might be a key step for controlling endocytosis, and thus aid in homeostasis and cell physiology.
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. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.
Sorting of ubiquitinated endosomal membrane proteins into the MVB pathway is executed by the class E Vps protein complexes ESCRT-I, -II, and -III, and the AAA-type ATPase Vps4. This study ...characterizes ESCRT-II, a soluble ∼155 kDa protein complex formed by the class E Vps proteins Vps22, Vps25, and Vps36. This protein complex transiently associates with the endosomal membrane and thereby initiates the formation of ESCRT-III, a membrane-associated protein complex that functions immediately downstream of ESCRT-II during sorting of MVB cargo. ESCRT-II in turn functions downstream of ESCRT-I, a protein complex that binds to ubiquitinated endosomal cargo. We propose that the ESCRT complexes perform a coordinated cascade of events to select and sort MVB cargoes for delivery to the lumen of the vacuole/lysosome.
Endocytosis of receptors at the plasma membrane is controlled by a complex mechanism that includes clathrin, adaptors, and actin regulators. Many of these proteins are conserved in yeast yet lack ...observable mutant phenotypes, which suggests that yeast endocytosis may be subject to different regulatory mechanisms. Here, we have systematically defined genes required for internalization using a quantitative genome-wide screen that monitors localization of the yeast vesicle-associated membrane protein (VAMP)/synaptobrevin homologue Snc1. Genetic interaction mapping was used to place these genes into functional modules containing known and novel endocytic regulators, and cargo selectivity was evaluated by an array-based comparative analysis. We demonstrate that clathrin and the yeast AP180 clathrin adaptor proteins have a cargo-specific role in Snc1 internalization. We additionally identify low dye binding 17 (LDB17) as a novel conserved component of the endocytic machinery. Ldb17 is recruited to cortical actin patches before actin polymerization and regulates normal coat dynamics and actin assembly. Our findings highlight the conserved machinery and reveal novel mechanisms that underlie endocytic internalization.
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. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μ-homology domain interacts with the Bmp receptor Alk8, uncovering an endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus challenge the primacy of FCHO1/2 in coat initiation.
Aguilar and Wendland discuss two studies that show how Ub can act as an endocytic signal in mammalian cells; in the absence of clathrin, Ub can direct entry via a CIE pathway. It appears that ...endocytic factors may play multiple roles, depending on which signal the cargo protein presents, or which endocytic route is being used.
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