The Saccharomyces cerevisiae APS1 gene encodes a homolog of the small subunit of the mammalian clathrin AP-1 complex: evidence for functional interaction with clathrin at the Golgi complex.

Abstract

Clathrin‐associated protein (AP) complexes have been implicated in the assembly of clathrin coats and the selectivity of clathrin‐mediated protein transport processes. We have identified a yeast gene, APS1, encoding a homolog of the small (referred to herein as sigma) subunits of the mammalian AP‐1 complex. Sequence comparisons have shown that Aps1p is more similar to the sigma subunit of the Golgi‐localized mammalian AP‐1 complex than Aps2p, which is more related to the plasma membrane AP‐2 sigma subunit. Like their mammalian counterparts, Aps1p and Aps2p are components of distinct, large (> 200 kDa) complexes and a significant portion of the Aps proteins co‐fractionate with clathrin‐coated vesicles during gel filtration chromatography. Unexpectedly, even though the evolutionary conservation of AP small subunits is substantial (50% identity between mammalian and yeast proteins), disruptions of APS1 (aps1 delta) and APS2 (aps2 delta), individually or in combination, elicit no detectable mutant phenotypes. These data indicate that the Aps proteins are not absolutely required for clathrin‐mediated selective protein transport in cells expressing wild type clathrin. However, aps1 delta accentuated the slow growth and alpha‐factor pheromone maturation defect of cells carrying a temperature‐sensitive allele of clathrin heavy chain (Chc) (chc1‐ts). In contrast, aps1 delta did not influence the effects of chc1‐ts on vacuolar protein sorting or receptor‐mediated endocytosis. The aps2 delta mutation resulted in a slight effect on chc1‐ts cell growth but had no additional effects. The growth defect of cells completely lacking Chc was compounded by aps1 delta but not aps2 delta. These results comprise evidence that Aps1p is involved in a subset of clathrin functions at the Golgi apparatus. The effect of aps1 delta on cells devoid of clathrin function suggests that Aps1p also participates in clathrin‐independent processes.

The Light Chain Subunit Is Required for Clathrin Function in Saccharomyces cerevisiae

“…7A). A portion of Kex2p also coeluted with Chc1p and Aps1p, consistent with a role for clathrin coats in the localization of Kex2p to the TGN as reported previously (21). Electron microscopy of samples negatively stained with uranyl acetate confirmed the presence of clathrin-coated vesicles in these fractions (data not shown).…”

Characterization of a Fourth Adaptor-related Protein Complex

“…Further insights into evolutionary relationships can be obtained by looking for homologues of the AP subunits in lower eukaryotes. In the budding yeast S. cerevisiae, in which the entire genome is known, genes have been identified that encode subunits sufficient to form three complete AP complexes, and there is strong genetic and biochemical evidence that yeast has AP-1 and AP-3 complexes that are functionally equivalent to AP-1 and AP-3 in mammals (Phan et al, 1994;Rad et al, 1995;Stepp et al, 1995;Cowles et al, 1997;Panek et al, 1997). It has been proposed that yeast also has an AP-2 complex, because the remaining AP subunits in yeast are more homologous to mammalian AP-2 subunits than to the subunits of any of the other AP complexes , including AP-4.…”

Composite synthetic lethal identification of membrane traffic inhibitors

“…At time points indicated for Ste3p and at 30 min of chase for ␣-factor maturation, 0.5 OD 600 in 200 l were transferred to tubes containing sodium fluoride and sodium azide (10 mM final). Immunoprecipitation of ␣-factor and cell lysis and immunoprecipitation of Ste3p were performed as described (31,32). For pulse-chase immunoprecipitations of ALP, CPS, and ␣-factor, cells were treated as above, except that in the case of ALP cells were labeled for 5 min and in all cases the chase was initiated by addition of 1:10 volume of 2% yeast extract, 0.3% cysteine, and methionine.…”