COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP

Abstract

We performed a systematic mapping of interaction domains on COP I subunits to gain novel insights into the architecture of coatomer. Using the two-hybrid system, we characterize the domain structure of the a-, b ¢-, e-COP and b-, g-, d-, z-COP coatomer subcomplexes and identify links between them that contribute to coatomer integrity. Our results demonstrate that the domain organization of the b-, g-, d-, z-COP subcomplex and AP adaptor complexes is related. Through in vivo analysis of a-COP truncation mutants, we characterize distinct functional domains on a-COP. Its N-terminal WD40 domain is dispensable for yeast cell viability and overall coatomer function, but is required for KKXX-dependent traf®cking. The last~170 amino acids of a-COP are also nonessential for cell viability, but required for e-COP incorporation into coatomer and maintainance of normal e-COP levels. Further, we demonstrate novel direct interactions of coatomer subunits with regulatory proteins: b ¢-and g-COP interact with the ARF-GTP-activating protein (GAP) Glo3p, but not Gcs1p, and b-and e-COP interact with ARF-GTP. Glo3p also interacts with intact coatomer in vitro.

The α- and β′-COP WD40 Domains Mediate Cargo-selective Interactions with Distinct Di-lysine Motifs

“…We previously demonstrated, by using a truncation mutant lacking this domain, that the WD40 domain of ␣-COP is not required for yeast cell viability but is essential for KKTN-dependent trafficking and binding to the KKTN peptide in vitro (Eugster et al, 2000). Here, we investigate the function of the corresponding WD40 domain present on ␤Ј-COP.…”

“…Rabbit antisera used were anti-coatomer, anti-␣-COP, anti-␤Ј-COP ; anti-␥-COP (Hosobuchi et al, 1992); anti-⑀-COP (Eugster et al, 2000); anti-Sed5p (this study); and anti-Emp47p (Schrö der- Kohne et al, 1998). The ␣-COP fragment was specifically detected using an antiserum against a COOH-terminal ␣-COP peptide (residues 1155-1171 of Ret1p).…”

“…The LexA two-hybrid system (Estojak et al, 1995;Golemis et al, 1996) was used as described previously (Eugster et al, 2000). Yeast reporter strains EGY48 (Table 1), as well as the bait plasmids pEG202 and the prey plasmids pJG4-5 were used (Golemis et al, 1996).…”

“…SDS-PAGE, immunoblot analysis by using enhanced chemiluminescence radiolabeling of cells, immunoprecipitation, and PhosphorImager quantitation were as described previously (Duden et al, , 1998Eugster et al, 2000). Rabbit antisera used were anti-coatomer, anti-␣-COP, anti-␤Ј-COP ; anti-␥-COP (Hosobuchi et al, 1992); anti-⑀-COP (Eugster et al, 2000); anti-Sed5p (this study); and anti-Emp47p (Schrö der- Kohne et al, 1998).…”

Systematic Structure-Function Analysis of the Small GTPase Arf1 in Yeast

“…This, in turn, has been proposed to result in one or both of the following effects in cells: a shift toward the GTP-bound conformation and interaction with effector proteins or a shift toward nucleotide-free protein and subsequent sequestra-tion of GEFs (Feig et al, 1986;Ziman et al, 1991;Jones et al, 1995;Schmidt et al, 1996;Cool et al, 1999). However, in two-hybrid studies, Arf1-N126I (a mutation in the guanine specificity region) not only binds effector proteins coatomer subunits ␤ and ⑀ but also the GAP proteins Gcs1 and Glo3 (Eugster et al, 2000). This, combined with the observation that arf1-117 maps to the crystallographically determined ArfGAP-binding site (Goldberg, 1999), suggests that the lethality of arf1-118 may be due to aberrant binding to Arf-GAP.…”