[show abstract][hide abstract] ABSTRACT: The importance of coupling the process of endocytosis to factors that regulate actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified. Sla1p is a well-characterized yeast protein that binds both to activators of actin dynamics, Las17p and Pan1p, and to cargo proteins, such as the pheromone receptor Ste2p. Previously, we reported that the Lsb5 protein plays a role in endocytosis in yeast and that it localizes to the plasma membrane. Lsb5p has a similar structure to the GGA [Golgi-localized, gamma-ear-containing, Arf (ADP-ribosylation factor)-binding] family of proteins with an N-terminal VHS [Vps27p (vacuolar protein sorting protein 27), Hrs, Stam] domain and a GAT (GGA and Tom1) domain. It does not, however, contain either a gamma-adaptin ear or a clathrin-binding motif. In the present study, we have further defined its interaction site with both Sla1p and with Las17p, two regulators of actin dynamics. The site of interaction with Sla1p involves the Sla1 HD1 (homology domain 1), which also was shown previously to interact with the pheromone receptor Ste2p. We also demonstrate hitherto unknown interactions between Lsb5p and the active form of the yeast Arf3 protein, and with ubiquitin. Finally, we demonstrate a requirement for Arf3p expression in order to localize Lsb5p to the correct cortical site in cells. Taken together, our data provide further evidence for the role of Lsb5p in membrane-trafficking events at the plasma membrane and also demonstrate for the first time an interaction of Arf3 with the endocytic machinery in yeast.
[show abstract][hide abstract] ABSTRACT: The importance of a dynamic actin cytoskeleton for facilitating endocytosis has been recognised for many years in budding yeast and is increasingly recognised in mammalian cells. However, the mechanism for actin recruitment and the role it plays in endocytosis is unclear. Here we show the importance of two yeast proteins in this process. We demonstrate that Sla1p and Sla2p interact in vitro and in vivo and that this interaction is mediated by the central domain of Sla2p, which includes its coiled-coil region, and by a domain of Sla1p between residues 118 and 361. Overexpression of the interacting fragment of Sla1p causes reduced fluid-phase endocytosis and, interestingly, defects in subsequent trafficking to vacuoles. We show that Sla2p is required for the polarised localisation of Sla1p in cells but not for its cortical localisation or for its overlapping localisation with actin. Generation of an Deltasla1Deltasla2 double mutant demonstrates that Sla2p is likely to act upstream of Sla1p in endocytosis, whereas sensitivity to latrunculin-A suggests that the proteins have opposite effects on actin dynamics. We propose that Sla2p recruits Sla1p to endocytic sites. Sla1p and its associated protein Pan1p then regulate actin assembly through interactions with Arp2/3 and Arp2/3-activating proteins Abp1p and Las17/Bee1p.
[show abstract][hide abstract] ABSTRACT: The importance of coupling the process of endocytosis to factors regulating actin dynamics has been clearly demonstrated in yeast, and many proteins involved in these mechanisms have been identified and characterized. Here we demonstrate the importance of two additional cortical components, Ysc84p and Lsb5p, which together are essential for the organization of the actin cytoskeleton and for fluid phase endocytosis. Both Ysc84p and Lsb5p were identified through two-hybrid screens with different domains of the adaptor protein Sla1p. Ysc84p colocalizes with cortical actin and requires the presence of an intact actin cytoskeleton for its cortical localization. Ycl034w/Lsb5p localizes to the cell cortex but does not colocalize with actin. The Lsb5 protein contains putative VHS and GAT domains as well as an NPF motif, which are all domains characteristic of proteins involved in membrane trafficking. Deletion of either gene alone does not confer any dramatic phenotype on cells. However, deletion of both genes is lethal at elevated temperatures. Furthermore, at all temperatures this double mutant has depolarized actin and an almost undetectable level of fluid phase endocytosis. Our data demonstrate that Ysc84p and Lsb5p are important components of complexes involved in overlapping pathways coupling endocytosis with the actin cytoskeleton in yeast.
Molecular Biology of the Cell 11/2002; 13(10):3646-61. · 4.60 Impact Factor
[show abstract][hide abstract] ABSTRACT: Sla1p is a protein required for cortical actin patch structure and organisation in budding yeast. Here we use a combination of immunofluorescence microscopy and biochemical approaches to demonstrate interactions of Sla1p both with proteins regulating actin dynamics and with proteins required for endocytosis. Using Sla1p-binding studies we reveal association of Sla1p with two proteins known to be important for activation of the Arp2/3 complex in yeast, Abp1p and the yeast WASP homologue Las17p/Bee1p. A recent report of Sla1p association with Pan1p puts Sla1p in the currently unique position of being the only yeast protein known to interact with all three known Arp2/3-activating proteins in yeast. Localisation of Sla1p at the cell cortex is, however, dependent on the EH-domain-containing protein End3p, which is part of the yeast endocytic machinery. Using spectral variants of GFP on Sla1p (YFP) and on Abp1p (CFP) we show for the first time that these proteins can exist in discrete complexes at the cell cortex. However, the detection of a significant FRET signal means that these proteins also come close together in a single complex, and it is in this larger complex that we propose that Sla1p binding to Abp1p and Las17p/Bee1p is able to link actin dynamics to the endocytic machinery. Finally, we demonstrate marked defects in both fluid-phase and receptor-mediated endocytosis in cells that do not express SLA1, indicating that Sla1p is central to the requirement in yeast to couple endocytosis with the actin cytoskeleton.