The trans-Golgi network GRIP-domain proteins form alpha-helical homodimers.
A recently described family of TGN (trans-Golgi network) proteins, all of which contain a GRIP domain targeting sequence, has been proposed to play a role in membrane transport. On the basis of the high content of heptad repeats, GRIP domain proteins are predicted to contain extensive coiled-coil regions that have the potential to mediate protein-protein interactions. Four mammalian GRIP domain proteins have been identified which are targeted to the TGN through their GRIP domains, namely p230, golgin-97, GCC88 and GCC185. In the present study, we have investigated the ability of the four mammalian GRIP domain proteins to interact. Using a combination of immunoprecipitation experiments of epitope-tagged GRIP domain proteins, cross-linking experiments and yeast two-hybrid interactions, we have established that the GRIP proteins can self-associate to form homodimers exclusively. Two-hybrid analysis indicated that the N- and C-terminal fragments of GCC88 can interact with themselves but not with each other, suggesting that the GRIP domain proteins form parallel coiled-coil dimers. Analysis of purified recombinant golgin-97 by CD spectroscopy indicated a 67% alpha-helical structure, consistent with a high content of coiled-coil sequences. These results support a model for GRIP domain proteins as extended rod-like homodimeric molecules. The formation of homodimers, but not heterodimers, indicates that each of the four mammalian TGN golgins has the potential to function independently.
Luke
Michael R
MR
The Russell Grimwade School of Biochemistry and Molecular Biology and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Melbourne, VIC 3010, Australia.
Houghton
Fiona
F
Perugini
Matthew A
MA
Gleeson
Paul A
PA
D000818:Animals; D001324:Autoantigens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019556:COS Cells; D002522:Chlorocebus aethiops; D002942:Circular Dichroism; D019281:Dimerization; D000077614:Golgi Matrix Proteins; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D021601:trans-Golgi Network / Q000378:metabolism*
D001324:Autoantigens; C472298:GCC1 protein, human; C472299:GCC2 protein, human; C098772:GOLGA4 protein, human; D000077614:Golgi Matrix Proteins; C450721:Golgi complex autoantigen, 97-kDa; D008565:Membrane Proteins; D026901:Membrane Transport Proteins