The yeast actin-related protein Arp2p is required for the internalization step of endocytosis.

Abstract

The Saccharomyces cerevisiae actin-related protein Arp2p is an essential component of the actin cytoskeleton. We have tested its potential role in the endocytic and exocytic pathways by using a temperature-sensitive allele, arp2-1. The fate of the plasma membrane transporter uracil permease was followed to determine whether Arp2p plays a role in the endocytic pathway. Inhibition of normal endocytosis as revealed by maintenance of active uracil permease at the plasma membrane and strong protection against subsequent vacuolar degradation of the protein were observed in the mutant at the restrictive temperature. Furthermore, arp2-1 cells accumulated ubiquitin-permease conjugates, formed prior to internalization. These effects were also visible at permissive temperature, whereas the actin cytoskeleton appeared to be normally polarized. The soluble hydrolase carboxypeptidase Y and the lipophilic dye FM 4-64 were targeted normally to the vacuole in arp2-1 cells. Thus, Arp2p is required for internalization but does not play a major role in later steps of endocytosis. Synthetic lethality was demonstrated between arp2-1 and the endocytic mutant end3-1, suggesting participation of Arp2p and End3p in the same process. Finally, no evidence for a major defect in secretion was apparent; invertase secretion and delivery of uracil permease to the plasma membrane were unaffected in arp2-1 cells.

ARPC1/Arc40 Mediates the Interaction of the Actin-related Protein 2 and 3 Complex with Wiskott-Aldrich Syndrome Protein Family Activators

“…The anti-yeast Arp2 polyclonal antibody was raised in rabbits against an Arp2 peptide as described previously (14). The anti-yeast ARPC1/Arc40 antibody (YAR40) was raised in rabbits as described previously (10).…”

Ubiquitin–Proteasome-dependent Degradation of a Mitofusin, a Critical Regulator of Mitochondrial Fusion

“…After addition of 1 ml chase media (SD-Met supplemented with 6 mg/ml methionine and 2 mg/ml BSA), cells were incubated at 37°C. After addition of chase media ϳ2 ODs of cells were collected immediately (time ϭ 0) and at 30 and 60 min and treated as described previously (Moreau et al, 1997). Results were quantified by Storm Phosphorimager and ImageQuant software (GE Healthcare, Waukesha, WI).…”

Endocytosis and vacuolar morphology inSaccharomyces cerevisiae are altered in response to ethanol stress or heat shock

“…Vida and Emr (1995) concluded from their studies that FM 4-64 would be a useful tool in the analysis of endocytosis and vacuolar dynamics in yeast. Subsequent reports have borne this out (Gaynor et al, 1998;Hill et al, 1996;Luo and Chang, 1997;Moreau et al, 1997;Rieder et al, 1996;Rieder and Emr, 1997;Roeder and Shaw, 1996;Wang et al, 1996). Fluorescence-activated cell sorting of yeast cells defective in the internalization of FM 4-64 has also been successfully used to identify novel endocytic mutants (Wendland et al, 1996;Zheng et al, 1998).…”