Adaptor Complex-independent Clathrin Function in Yeast

Abstract

Clathrin-associated adaptor protein (AP) complexes are major structural components of clathrin-coated vesicles, functioning in clathrin coat assembly and cargo selection. We have carried out a systematic biochemical and genetic characterization of AP complexes in Saccharomyces cerevisiae. Using coimmunoprecipitation, the subunit composition of two complexes, AP-1 and AP-2R, has been defined. These results allow assignment of the 13 potential AP subunits encoded in the yeast genome to three AP complexes. As assessed by in vitro binding assays and coimmunoprecipitation, only AP-1 interacts with clathrin. Individual or combined disruption of AP-1 subunit genes in cells expressing a temperature-sensitive clathrin heavy chain results in accentuated growth and alpha-factor pheromone maturation defects, providing further evidence that AP-1 is a clathrin adaptor complex. However, in cells expressing wild-type clathrin, the same AP subunit deletions have no effect on growth or alpha-factor maturation. Furthermore, gel filtration chromatography revealed normal elution patterns of clathrin-coated vesicles in cells lacking AP-1. Similarly, combined deletion of genes encoding the beta subunits of the three AP complexes did not produce defects in clathrin-dependent sorting in the endocytic and vacuolar pathways or alterations in gel filtration profiles of clathrin-coated vesicles. We conclude that AP complexes are dispensable for clathrin function in S. cerevisiae under normal conditions. Our results suggest that alternative factors assume key roles in stimulating clathrin coat assembly and cargo selection during clathrin-mediated vesicle formation in yeast.

A Role For Lte1p (a Low Temperature Essential Protein Involved in Mitosis) in Proprotein Processing in the Yeast Secretory Pathway

“…6). These results are compatible with a growing list of mutants in trafficking genes that affect Golgi-early endosomal trafficking and secretory proprotein processing yet do not produce a vps phenotype, such as tlg2 (and to a large extent, tlg1 (25), chc1 (26), as well as inp53 (27,28)). As one intracellular site of Vps21p function involves Golgi-early endosome trafficking (29), Lte1p may facilitate Vps21p activity at this site; thus lte1 should be considered a candidate for addition to the foregoing list of mutants.…”

The Rsp5 Ubiquitin Ligase Binds to and Ubiquitinates Members of the Yeast CIN85-Endophilin Complex, Sla1-Rvs167

“…Unlike the amphiphysins and endophilins, Rvs167 does not carry clathrin and clathrin adaptor (AP)-binding motifs and does not appear to bind either of these proteins. This difference may exist because clathrin adaptors are not required for receptor internalization in yeast (61,62), and clathrin itself is not strictly required (63).…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…AP-2 is central to the development of mammals and Drosophila because disruption of the subunit genes leads to lethality (González-Gaitán and Jäckle, 1997; Mitsunari et al, 2005). By contrast, the budding yeast Saccharomyces cerevisiae lacking all three AP b-subunit genes, including the AP-2 homologs, exhibits no deleterious effects on cell growth (Yeung et al, 1999). The only role for the budding yeast AP-2 reported to date is in endocytosis of the killer toxin K28, whereas the internalization of other cargo proteins appears to be independent of AP-2 (Carroll et al, 2009).…”