Search

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.

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.

Slalp is a protein required for cortical actin cytoskeleton structure and organisation in the budding yeast Saccharomyces cerevisiae. Slal null cells display aberrant cortical actin patches which are fewer in number but significantly enlarged. Movement of these patches is also significantly reduced in slal null cells. Expression of mutant forms of Slalp in which distinct domains within its primary sequence are deleted has shown that phenotypes associated with the full deletion are functionally separable. In particular, the C-terminal repeat region is required if cells are to grow in the absence of cortical patch protein Abplp and a region encompassing the third of its three SH3 domains is important for the highly polarised and punctate appearance of cortical actin patches in wildtype cells. To investigate the role of Slalp in actin dynamics further an slal mutant lacking the third SH3 domain has been integrated into the yeast genome (slal-Delta118-511) and expressed as the sole form of Slalp in cells. Rhodamine-phalloidin staining of this mutant showed aberrant cortical actin patches similar to the slal null phenotype. Both myc and GFP tagged mutant proteins localise to the cell cortex but do not show any co-localisation with the aberrant actin patches, implicating this domain in actin cytoskeleton interaction but showing it to be unnecessary for cortical localisation. A role for Slalp in enhancing actin turnover is also suggested by the observation that yeast expressing the mutant Slalp exhibit a significantly reduced sensitivity to Latrunculin compared to wild-type cells (indicating increased F-actin stability). A two-hybrid screen using the slal-118-511 domain as bait identified two actin- associated proteins as binding partners: Sla2p and Ysc84p. Sla2p was identified in the same synthetic-lethal screen as Slalp and is required for a normal actin cytoskeleton and endocytosis. Ysc84p is a novel protein that requires Abp1p (but not Slalp) for localisation to cortic