SummaryDynamins are a conserved family of proteins involved in membrane fusion and fission. Although mammalian dynamins are known to be involved in several membrane-trafficking events, the role of dynamin-1 in endocytosis is the best-characterised role of this protein family. Despite many similarities between endocytosis in yeast and mammalian cells, a comparable role for dynamins in yeast has not previously been demonstrated. The reported lack of involvement of dynamins in yeast endocytosis has raised questions over the general applicability of the current yeast model of endocytosis, and has also precluded studies using well-developed methods in yeast, to further our understanding of the mechanism of dynamin function during endocytosis. Here, we investigate the yeast dynamin-like protein Vps1 and demonstrate a transient burst of localisation to sites of endocytosis. Using live-cell imaging of endocytic reporters in strains lacking vps1, and also electron microscopy and biochemical approaches, we demonstrate a role for Vps1 in facilitating endocytic invagination. Vps1 mutants were generated, and analysis in several assays reveals a role for the C-terminal self-assembly domain in endocytosis but not in other membrane fission events with which Vps1 has previously been associated.
Dynamins are a conserved family of proteins involved in many membrane fusion and fission events. Previously, the dynamin-related protein Vps1 was shown to localize to endocytic sites, and yeast carrying deletions for genes encoding both the BAR domain protein Rvs167 and Vps1 had a more severe endocytic scission defect than either deletion alone. Vps1 and Rvs167 localize to endocytic sites at the onset of invagination and disassemble concomitant with inward vesicle movement. Endocytosis is a highly regulated eukaryotic process in which small regions of the plasma membrane invaginate into the cell and are pinched off, resulting in a vesicle that is then able to fuse with endosomes and enter the endolysosomal system. The process is required for remodelling the surface of cells to ensure the appropriate complement of receptors and transporters are exposed.In yeast, more than 50 different proteins are involved in endocytosis, and many of these proteins assemble and disassemble in a transient and highly choreographed way at endocytic sites on the plasma membrane (1,2) (reviewed in 3). Careful analysis of the behaviour of proteins at the sites has allowed models to be generated that indicate distinct endocytic stages. Early stages are associated with localization of clathrin and other endocytic patch coat proteins, while later stages involve the actindriven invagination of the membrane, followed by scission to form a vesicle.
The yeast SM22 homologue Scp1 has previously been shown to act as an actin-bundling protein in vitro. In cells, Scp1 localizes to the cortical actin patches that form as part of the invagination process during endocytosis, and its function overlaps with that of the well characterized yeast fimbrin homologue Sac6p. In this work we have used live cell imaging to demonstrate the importance of key residues in the Scp1 actin interface. We have defined two actin binding domains within Scp1 that allow the protein to both bind and bundle actin without the need for dimerization. Green fluorescent protein-tagged mutants of Scp1 also indicate that actin localization does not require the putative phosphorylation site Ser-185 to be functional. Deletion of SCP1 has few discernable effects on cell growth and morphology. However, we reveal that scp1 deletion is compensated for by up-regulation of Sac6. Furthermore, Scp1 levels are increased in the absence of sac6. The presence of compensatory pathways to up-regulate Sac6 or Scp1 levels in the absence of the other suggest that maintenance of sufficient bundling activity is critical within the cell. Analysis of cortical patch assembly and movement during endocytosis reveals a previously undetected role for Scp1 in movement of patches away from the plasma membrane. Additionally, we observe a dramatic increase in patch lifetime in a strain lacking both sac6 and scp1, demonstrating the central role played by actin-bundling proteins in the endocytic process.
SummaryActin is critical for endocytosis in yeast cells, and also in mammalian cells under tension. However, questions remain as to how force generated through actin polymerization is transmitted to the plasma membrane to drive invagination and scission. Here, we reveal that the yeast dynamin Vps1 binds and bundles filamentous actin. Mutational analysis of Vps1 in a helix of the stalk domain identifies a mutant RR457-458EE that binds actin more weakly. In vivo analysis of Vps1 function demonstrates that the mutation disrupts endocytosis but not other functions of Vps1 such as vacuolar trafficking or peroxisome fission. The mutant Vps1 is stably expressed in cells and co-localizes with the endocytic reporters Abp1 and the amphiphysin Rvs167. Detailed analysis of individual endocytic patch behavior indicates that the mutation causes aberrant movements in later stages of endocytosis, consistent with a scission defect. Ultrastructural analysis of yeast cells using electron microscopy reveals a significant increase in invagination depth, further supporting a role for the Vps1-actin interaction during scission. In vitro analysis of the mutant protein demonstrates that—like wild-type Vps1—it is able to form oligomeric rings, but, critically, it has lost its ability to bundle actin filaments into higher-order structures. A model is proposed in which actin filaments bind Vps1 during invagination, and this interaction is important to transduce the force of actin polymerization to the membrane to drive successful scission.
The AP-2 complex is a heterotetrameric endocytic cargo-binding adaptor that facilitates uptake of membrane proteins during mammalian clathrin-mediated endocytosis. While budding yeast has clear homologues of all four AP-2 subunits which form a complex and localize to endocytic sites in vivo, the function of yeast AP-2 has remained enigmatic. Here, we demonstrate that AP-2 is required for hyphal growth in Candida albicans and polarized cell responses in Saccharomyces cerevisiae. Deletion of APM4, the cargo-binding mu subunit of AP-2, causes defects in pseudohyphal growth, generation of a mating projection and the cell wall damage response. In an apm4 null mutant, the cell wall stress sensor Mid2 is unable to relocalize to the tip of a mating projection following pheromone addition, or to the mother bud neck in response to cell wall damage. A direct binding interaction between Mid2 and the mu homology domain of Apm4 further supports a model in which AP-2 binds Mid2 to facilitate its internalization and relocalization in response to specific signals. Thus, Mid2 is the first cargo for AP-2 identified in yeast. We propose that endocytic recycling of Mid2 and other components is required for polarized cell responses ensuring cell wall deposition and is tightly monitored during cell growth.
Phosphatidylinositol-(4,5)-bisphosphate [PtdIns(4,5)P2] is a key regulator of endocytosis. PtdIns(4,5)P2 generation at the plasma membrane in yeast is mediated by the kinase Mss4p, but the mechanism underlying the temporal and spatial activation of Mss4p to increase formation of PtdIns(4,5)P2 at appropriate sites is not known. Here, we show that ADP ribosylation factor (Arf)3p, the yeast homologue of mammalian Arf6, is necessary for wildtype levels of PtdIns(4,5)P2 at the plasma membrane. Arf3p localizes to dynamic spots at the membrane, and the behaviour of these is consistent with it functioning in concert with endocytic machinery. Localization of Arf3p is disrupted by deletion of genes encoding an ArfGAP homology protein Gts1p and a guanine nucleotide exchange factor Yel1p. Significantly, deletion of arf3 causes a reduction in PtdIns(4,5)P2 at the plasma membrane, while increased levels of active Arf3p, caused by deletion of the GTPase-activating protein Gts1, increase PtdIns(4,5)P2 levels. Furthermore, elevated Arf3p correlates with an increase in the number of endocytic sites. Our data provide evidence for a mechanism in yeast to positively regulate plasma membrane production of PtdIns(4,5)P2 levels and that these changes impact on endocytosis.
The human fungal pathogen Candida albicans is known to require endocytosis to enable its adaptation to diverse niches and to maintain its highly polarized hyphal growth phase. While studies have identified changes in transcription leading to the synthesis and secretion of new proteins to facilitate hyphal growth, effective maintenance of hyphae also requires concomitant removal or relocalization of other cell surface molecules. The key molecules which must be removed from the cell surface, and the mechanisms behind this, have, however, remained elusive. In this study, we show that the AP-2 endocytic adaptor complex is required for the internalization of the major cell wall biosynthesis enzyme Chs3. We demonstrate that this interaction is mediated by the AP-2 mu subunit (Apm4) YXXΦ binding domain. We also show that in the absence of Chs3 recycling via AP-2, cells have abnormal cell wall composition, defective polarized cell wall deposition, and morphological defects. The study also highlights key distinctions between endocytic requirements of growth at yeast buds compared to that at hyphal tips and different requirements of AP-2 in maintaining the polarity of mannosylated proteins and ergosterol at hyphal tips. Together, our findings highlight the importance of correct cell wall deposition in cell shape maintenance and polarized growth and the key regulatory role of endocytic recycling via the AP-2 complex. IMPORTANCE Candida albicans is a human commensal yeast that can cause significant morbidity and mortality in immunocompromised individuals. Within humans, C. albicans can adopt different morphologies as yeast or filamentous hyphae and can occupy different niches with distinct temperatures, pHs, CO2 levels, and nutrient availability. Both morphological switching and growth in different environments require cell surface remodelling, which involves both the addition of newly synthesized proteins as well as the removal of other proteins. In our study, we demonstrate the importance of an adaptor complex AP-2 in internalizing and recycling a specific cell surface enzyme to maintain effective polarized hyphal growth. Defects in formation of the complex or in its ability to interact directly with cargo inhibit enzyme uptake and lead to defective cell walls and aberrant hyphal morphology. Our data indicate that the AP-2 adaptor plays a central role in regulating cell surface composition in Candida.
Clathrin-mediated endocytosis (CME) is a well characterized pathway in both yeast and mammalian cells. An increasing number of alternative endocytic pathways have now been described in mammalian cells that can be both clathrin, actin, and Arf6- dependent or independent. In yeast, a single clathrin-mediated pathway has been characterized in detail. However, disruption of this pathway in many mutant strains indicates that other uptake pathways might exist, at least for bulk lipid and fluid internalization. Using a combination of genetics and live cell imaging, here we show evidence for a novel endocytic pathway in S. cerevisiae that does not involve several of the proteins previously shown to be associated with the ‘classic’ pathway of endocytosis. This alternative pathway functions in the presence of low levels of the actin-disrupting drug latrunculin-A which inhibits movement of the proteins Sla1, Sla2, and Sac6, and is independent of dynamin function. We reveal that in the absence of the ‘classic’ pathway, the actin binding protein Abp1 is now essential for bulk endocytosis. This novel pathway appears to be distinct from another described alternative endocytic route in S. cerevisiae as it involves at least some proteins known to be associated with cortical actin patches rather than being mediated at formin-dependent endocytic sites. These data indicate that cells have the capacity to use overlapping sets of components to facilitate endocytosis under a range of conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.