Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex.
Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.
Faulstich
D
D
Institut für Biochemie I, Heidelberg, Germany.
Auerbach
S
S
Orci
L
L
Ravazzola
M
M
Wegchingel
S
S
Lottspeich
F
F
Stenbeck
G
G
Harter
C
C
Wieland
F T
FT
Tschochner
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain; D002417:Cattle; D003001:Cloning, Molecular; D018699:Coated Vesicles / Q000737:chemistry; D020756:Coatomer Protein; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D005804:Genes, Lethal / Q000235:genetics; D006056:Golgi Apparatus / Q000737:chemistry; D008099:Liver; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D020756:Coatomer Protein; D018076:DNA, Complementary; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins