guanosine-triphosphate and 2-3-dimethylmaleic-anhydride

COPI-coated vesicles mediate protein transport within the early secretory pathway. Their coat consists of ADP ribosylation factor (ARF1, a small guanosine nucleotide binding protein), and coatomer, a cytosolic complex composed of seven subunits, alpha- to zeta-coat proteins (COPs). For coat formation that initiates budding of a vesicle, ARF1 is recruited to the Golgi membrane from the cytosol in its GTP-bound form, and subsequently, coatomer can bind to the membrane. To identify a minimal structure of coatomer capable to bind to Golgi membranes in an ARF1-dependent manner, we have established a procedure to dissociate coatomer under conditions that allow reassociation of the subunits to a complete and functional complex. After dissociation, subunits or subcomplexes can be isolated and may be expected to be functional. Herein we describe isolation of a subcomplex of coatomer consisting of beta- and delta-COPs that is able to bind to Golgi membranes in an ARF1- and GTP-dependent manner.

Topics: ADP-Ribosylation Factor 1; ADP-Ribosylation Factors; Animals; Coatomer Protein; Cytosol; Dimerization; Golgi Apparatus; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Diphosphate; Guanosine Triphosphate; Liver; Macromolecular Substances; Maleic Anhydrides; Membrane Proteins; Microtubule-Associated Proteins; Rabbits; Thionucleotides

We have recently shown that rat liver 60 S ribosomal subunits active in protein synthesis can be reconstituted from inactive core particles lacking 30% of the total proteins, mainly L10a, L12, L22, L24, A33 and the acidic phosphoproteins P1-P2, obtained by treatment of 60 S subunits with dimethylmaleic anhydride [(1987) Eur. J. Biochem. 163, 15-20]. In this study, an ethanol extract of the 60 S subunit which contains only P1 P2 was also shown to be effective in reconstitution with the DMMA-core-particles: it strongly stimulated the EF-2-dependent GTP hydrolysis and, to a lesser extent, polyphenylalanine synthesis; like the DMMA wash it shifted the thermal denaturation curve of the DMMA-core particles towards that of control subunits. Prior dephosphorylation of the ethanol extract by alkaline phosphatase inhibited the reconstruction process.

Topics: Animals; Guanosine Triphosphate; Liver; Maleic Anhydrides; Peptide Elongation Factor 2; Peptide Elongation Factors; Phosphoproteins; Phosphorylation; Protein Biosynthesis; Rats; Ribosomal Proteins; Ribosomes