A giant protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins forms a cytosolic ternary complex with clathrin and Hsp70.
We have recently identified an unusually large human protein that stimulates guanine nucleotide exchange on ARF1 and Rab proteins (EMBO J 15: 4262-4273, 1996). This protein, designated p532 based on its predicted molecular weight (EMBO J 15: 5738, 1996), contains multiple structural domains including two regions of seven internal repeats highly related to the cell cycle regulator RCC1, a guanine nucleotide exchange factor for the small GTP-binding protein Ran, seven beta-repeat domains characteristic of the beta subunit of heterotrimeric G proteins, three putative SH3 binding sites, a putative leucine-zipper and a carboxy-terminal HECT domain characteristic of E3 ubiquitin-protein ligases. Some of these domains are known to be involved in protein-protein interactions, suggesting the existence of p532-interacting proteins. To identify some of these proteins, we used the carboxy-terminal RCC1-like domain (RLD) of p532 in the yeast two-hybrid system. We report here the isolation of a clone that encodes the last 654 amino acid residues of the clathrin heavy chain. This interaction involves amino acid residues 1315-1557 of the clathrin heavy chain and the carboxy, but not the amino-terminal RLD of p532. p532 has been located in the cytosolic fraction as well as in the Golgi apparatus. The interaction between p532 and clathrin only occurs in the cytosol and is mediated by the formation of an ATP-dependent ternary complex with the heat shock protein, Hsp70. These observations suggest that p532 is involved in membrane transport processes.
Rosa
J L
JL
Department of Molecular Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543, USA.
Barbacid
M
M
D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; D001665:Binding Sites; D018797:Cell Cycle Proteins; D002966:Clathrin / Q000737:chemistry*; D003600:Cytosol / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000737:chemistry*; D020662:Guanine Nucleotide Exchange Factors; D018840:HSP70 Heat-Shock Proteins; D006801:Humans; D009687:Nuclear Proteins; D011506:Proteins / Q000737:chemistry*; D014407:Tumor Cells, Cultured; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D002966:Clathrin; D004268:DNA-Binding Proteins; D020662:Guanine Nucleotide Exchange Factors; D018840:HSP70 Heat-Shock Proteins; D009687:Nuclear Proteins; D011506:Proteins; C062521:RCC1 protein, human; C102169:HERC1 protein, human; D044767:Ubiquitin-Protein Ligases; D019204:GTP-Binding Proteins; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors