End4p/Sla2p Interacts with Actin-associated Proteins for Endocytosis inSaccharomyces cerevisiae

Abstract

end4-1 was isolated as a temperature-sensitive endocytosis mutant. We cloned and sequenced END4 and found that it is identical to SLA2/MOP2. This gene is required for growth at high temperature, viability in the absence of Abp1p, polarization of the cortical actin cytoskeleton, and endocytosis. We used a mutational analysis of END4 to correlate in vivo functions with regions of End4p and we found that two regions of End4p participate in endocytosis but that the talin-like domain of End4p is dispensable. The N-terminal domain of End4p is required for growth at high temperature, endocytosis, and actin organization. A central coiled-coil domain of End4p is necessary for formation of a soluble sedimentable complex. Furthermore, this domain has an endocytic function that is redundant with the function(s) of ABP1 and SRV2. The endocytic function of Abp1p depends on its SH3 domain. In addition we have isolated a recessive negative allele of SRV2 that is defective for endocytosis. Combined biochemical, functional, and genetic analysis lead us to propose that End4p may mediate endocytosis through interaction with other actin-associated proteins, perhaps Rvs167p, a protein essential for endocytosis.

RNAi-mediated Hip1R Silencing Results in Stable Association between the Endocytic Machinery and the Actin Assembly Machinery

“…It has previously been reported that cells lacking Hip1 are, like yeast sla2 mutants, defective for endocytosis (Wesp et al, 1997;Metzler et al, 2003). We confirmed that A2 cells, expressing Hip1R at Ͻ10% of normal levels, also show a defect in transferrin internalization (Figure 2, A-C).…”

“…Deletion of SLA2, the budding yeast gene that encodes a Hip1R homologue, results in severe defects in actin organization, cell morphology, and endocytosis (Holtzman et al, 1993;Wesp et al, 1997;Yang et al, 1999). We therefore expected similar effects for A2 cells, which expressed Hip1R at the lowest levels.…”

“…Hip1R belongs to a conserved family of proteins that includes yeast Sla2p/End4p and mammalian Hip1. In yeast, Sla2p is absolutely required for endocytosis, for normal cell morphology and for a functional actin cytoskeleton (Holtzman et al, 1993;Wesp et al, 1997;Yang et al, 1999). Hip1 is closely related to Hip1R (ϳ50% overall amino acid sequence identity) and has been implicated in the pathology of Huntington disease (Kalchman et al, 1997;Wanker et al, 1997).…”

Defects in Structural Integrity of Ergosterol and the Cdc50p-Drs2p Putative Phospholipid Translocase Cause Accumulation of Endocytic Membranes, onto Which Actin Patches Are Assembled in Yeast

“…The cdc50⌬ mutation exhibited synthetic lethality with ric1⌬ and a temperature-sensitive ypt6 mutation as well as rgp1 (our unpublished results), consistent with involvement of Cdc50p and Drs2p in endocytic recycling (Hua et al, 2002;Saito et al, 2004). Srv2p is also involved in endocytosis as a component of cortical actin patches (Wesp et al, 1997). Vps1p, a dynamin in yeast, has been implicated in clathrindependent protein transport at the TGN (Bensen et al, 2000;Gurunathan et al, 2002).…”

“…This phenotype was specific to the cdc50⌬ erg3⌬ mutant and was not observed in actin-related mutants. Mutants of cortical actin patch assembly, sla2⌬ (Wesp et al, 1997) and myo3⌬ myo5-1 (Geli and Riezman, 1996), and actin cable assembly, tpm1-2 tpm2⌬ (Pruyne et al, 1998), assembled actin patches only at cortical sites (Figure 4, A and B), although, in the sla2⌬ mutant, some actin patches were localized at a short distance from the plasma membrane as described (Kaksonen et al, 2003). These results suggest that the actin cytoskeleton of the cdc50⌬ erg3⌬ mutant is disorganized in a fundamentally different manner from that of the known actin patch-or actin cable-deficient mutants.…”