Apm4, the mu subunit of yeast AP-2 interacts with Pkc1, and mutation of the Pkc1 consensus phosphorylation site Thr176 inhibits AP-2 recruitment to endocytic sites

Abstract

The AP-2 endocytic adaptor has been extensively characterized in mammalian cells and is considered to play a role both in cargo binding and in formation of endocytic sites. However, despite our detailed knowledge of mechanistic aspects of endocytic complex assembly and disassembly in the model organism Saccharomyces cerevisiae, no function of AP-2 had been described in wild-type yeast under normal growth conditions. A recent study however revealed that disruption of the complex caused by deletion of the gene encoding its mu subunit (APM4) caused defects in cell polarity such that responses to pheromone, nutritional status and cell wall damage were affected. Furthermore, a homozygous deletion of the mu subunit gene in Candida albicans affected its ability to grow hyphae. Direct binding to the yeast cell wall stress sensor Mid2 was detected, and in an apm4 deletion strain Mid2 showed reduced re-localization to the mother bud neck region following cell wall damage with calcofluor or to the mating projection tip. Here we demonstrate an interaction between Apm4 and the yeast cell wall integrity pathway component Pkc1 and show that mutation of the predicted Pkc1 site in the Apm4 hinge region affects recruitment of the AP-2 complex to endocytic sites.

AP-2-Dependent Endocytic Recycling of the Chitin Synthase Chs3 Regulates Polarized Growth in Candida albicans

“…cerevisiae and in C. albicans; however, a functional role of mu subunit modifications has yet to be demonstrated in fungi (52–54).…”

The TORC2‐Dependent Signaling Network in the Yeast Saccharomyces cerevisiae

“…Also, contrary to the findings implicating Syp1 in septin ring disassembly and in endocytic actin patch function, is a study claiming that Syp1 is phosphorylated in a Rho1- and Pkc1-dependent manner to promote septin collar assembly [ 242 ]. The context around only one of the two sites reportedly phosphorylated by Pkc1 in Syp1 resembles well the pseudosubstrate sequence in Pkc1 and other likely Pkc1 sites, whereas a putative Pkc1 site in the cargo-binding clathrin-associated adaptor Apm4 is a better match [ 237 ] ( Figure 7 ).…”

“… Reported sites of Pkc1 phosphorylation. The evidence for Pkc1 phosphorylation of the indicated site (red) in the gene products listed (position of first residue in the sequence shown indicated on the left) can be found described in the following citations: Amp4 [ 237 ]; Bck1 [ 215 , 238 ]; Cdc55 [ 239 ]; Hht1 and Hht2 [ 240 ]; Igo2 [ 239 ]; Ndd1 [ 241 ]; Nem1 [ 233 ]; Pah1 [ 233 ]; Rtt109 [ 240 ]; Syp1 [ 242 ]; and, Ura7 [ 233 , 243 ]. …”

The AP‐2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast

“…In S. cerevisiae , Pkc1p is delocalized in apm4 Δ mutants (Chapa‐y‐Lazo and Ayscough, ; Chapa‐y‐Lazo et al ., ); as Pkc1p influences several aspects of cell wall synthesis (Levin, ), the absence of AP‐2 might produce defects in cell wall structure. A defective cell wall structure could influence the resistance to K28 toxin detected in S. cerevisiae AP‐2 mutants (Carroll et al ., ), because cell wall components are the primary toxin‐binding sites (Schmitt and Radler, ).…”

“…Endocytosis acts as a diffusion barrier to ensure the polarized localization of membrane lipids and polarity proteins; conversely, some polarity factors such as Cdc42p regulate the positioning of the endocytic machinery and certain steps of endocytosis (Valdez‐Taubas and Pelham, ; Marco et al ., ; Shivas et al ., ; Nishimura et al ., ). In S. cerevisiae , the apm4 Δ mutant exhibits delocalized Mid2, Pkc1 and Cdc42 proteins and shows defects in polarized growth (Chapa‐y‐Lazo and Ayscough, ). In Neurospora crassa and Candida albicans elimination of AP‐2 subunits leads to polarity defects and abnormal growth (Chapa‐y‐Lazo et al ., ).…”