Clathrin Interactions with C‐Terminal Regions of the Yeast AP‐1 β and γ Subunits are Important for AP‐1 Association with Clathrin Coats

Abstract

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae. Using GST-fusion chromatography, two clathrin-binding sites were defined in the beta1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the gamma subunit. When introduced into chromosomal genes, point mutations in the beta1 clathrin-binding motifs, or deletion of the gamma C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The beta1 mutations or the gamma truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when beta1 and gamma mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and alpha-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the beta1 and gamma subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo.

Clathrin Functions in the Absence of the Terminal Domain Binding Site for Adaptor-associated Clathrin-Box Motifs

“…There are many different TGN/endosomal adaptor proteins in yeast (AP-1, Gga1, and Gga2 and the epsin-related proteins Ent3 and Ent5), and disruption of these adaptor genes results in TGN/endosomal sorting phenotypes (Costaguta et al, 2001;Mullins and Bonifacino, 2001;Duncan et al, 2003;Friant et al, 2003;Eugster et al, 2004). However, in studies in which adaptor proteins containing CBM mutations were analyzed, no significant phenotypes were observed (Mullins and Bonifacino, 2001;Yeung and Payne, 2001). At least four endocytic adaptors have been studied in yeast-the epsin-related proteins Ent1 and Ent2 and the AP180 homologues Yap1801 and Yap1802-and all four possess C-terminal CBMs in which the carboxylic acid at the end of the polypeptide substitutes for the final polar residue in the motif.…”

“…Likewise, if the TDs containing chc1-box mutations only possess a lower affinity binding site for adaptor-associated CBMs, this low-affinity interaction between AP-1 and clathrin was easily disrupted during the cell fractionation procedure. In previous studies, AP-1 incorporation into CCVs was almost abolished in cells containing mutations to the two CBM sequences in the ␤-subunit (Apl2) and a C-terminal deletion of the clathrin-binding region in the ␥-subunit (Apl4) (Yeung and Payne, 2001). Thus, the reduced AP-1 association with CCVs isolated from chc1-box cells is consistent with these observations and suggests that stable incorporation of AP-1 into CCVs is mediated by the interaction of CBM-containing AP-1 subunits with the TD.…”

The Functional Relationship between the Cdc50p-Drs2p Putative Aminophospholipid Translocase and the Arf GAP Gcs1p in Vesicle Formation in the Retrieval Pathway from Yeast Early Endosomes to the TGN

“…In S. cerevisiae, there are three AP complexes, AP-1, AP-2R, and AP-3 (Yeung et al, 1999), and two GGAs, Gga1p and Gga2p (Costaguta et al, 2001;Dell'Angelica et al, 2000;Hirst et al, 2000). Both AP-1 and Gga1p/Gga2p physically interact with clathrin (Costaguta et al, 2001;Yeung and Payne, 2001;Yeung et al, 1999). It has been suggested that Gga1p and Gga2p are involved in the formation of CCVs in the TGN-to-late endosome transport pathway (Costaguta et al, 2001;Black and Pelham, 2000;Dell'Angelica et al, 2000;Hirst et al, 2000), although the interaction with Arf1p is not sufficient for the Golgi-localization and function of Gga1p/Gga2p (Boman et al, 2002).…”

Ternary Complex Formation of Ino2p-Ino4p Transcription Factors and Apl2p Adaptin β Subunit in Yeast

“…The C-terminal region contains two sequences related to the clathrin-binding motif [L (L, I) (D, E, N) (L, F) (D, E)] identified in mammalian clathrin-interacting proteins. 2,15) One sequence, LLDFD (638-642), precisely matches the mammalian consensus sequence, and the other, LLDLF, at the extreme Cterminus (722-726), is very similar to the motif except for one residue. In a previous study, Yeung and Payne reported that the C-terminal region of Apl2p (471-726) interacted with clathrin as detected by immunoblotting analysis.…”

“…14) The APL2 gene product, Apl2p, has been predicted to be a 726-amino acid protein and was shown to contain a clathrin-binding motif, LLDFD, at the C-terminal region (located at residues 722-726), 15,16) but the correlation between Apl2p and inositol biosynthesis has not been elucidated.…”