guanosine-triphosphate and metaperiodate

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.

Topics: Animals; Carrier Proteins; Cross-Linking Reagents; Dextrans; Dithiothreitol; Endoplasmic Reticulum, Rough; Ethylmaleimide; Guanosine Triphosphate; Magnesium; Membrane Fusion; Microscopy, Electron; Microsomes, Liver; N-Ethylmaleimide-Sensitive Proteins; Periodic Acid; Polyethylene Glycols; Rats; Serum Albumin, Bovine; Sulfhydryl Compounds; Sulfhydryl Reagents; Vesicular Transport Proteins; Zinc

We compared two approaches to identify and map small GTP-binding proteins in combination with high-resolution two-dimensional (2-D) gel electrophoresis. The first approach involved direct GTP ligand binding after a renaturing transfer onto nitrocellulose. In the second, affinity labeling with in situ periodate-oxidized GTP was used in permeabilized cells (Peter, M. E., She, J., Huber, L. A. and Terhorst, C. Anal. Biochem. 1993, 210, 77-85). Analysis by 2-D gel electrophoresis revealed a number of distinct intracellular small GTP-binding proteins in Madine-darby canine kidney strain II cells (MDCKII). Using specific antibodies the electrophoretic coordinates of rab4, rap1a/b, and rap2 were identified for native as well as for crosslinked GTPases. These methods allow the identification of small GTP-binding proteins in total cell lysates and purified subcellular fractions, providing excellent markers throughout the course of differentiation and development.

Topics: Animals; Cell Line; Electrophoresis, Gel, Two-Dimensional; GTP Phosphohydrolases; GTP-Binding Proteins; Guanosine Triphosphate; Oxidation-Reduction; Peptide Mapping; Periodic Acid

The amino acid sequence motifs of human c-H-ras p21 involved in the interaction with guanosine nucleotides were cross-linked to in situ periodate-oxidized [alpha-32P]GDP or [alpha-32P]GTP. Site-specific reaction was achieved by cross-linking conserved lysine residues close to the G-nucleotide binding site of p21 with the 2',3'-dialdehyde derivatives of GDP or GTP under kinetically controlled conditions. After endoproteinase Asp-N digestion, HPLC separation of 32P-labeled peptides and N-terminal microsequence analysis, two single lysine residues, namely, K117 and K147, which are parts of the N-K-X-D and S-A-K/L consensus elements of ras proteins, respectively, were identified. No significant divergences in the position and extent of covalent modification could be detected between p21.GDP and p21.GTP. This is in contrast to Thermus thermophilus EF-Tu.GDP and EF-Tu.GTP, which were investigated with the same technique [Peter, M. E., Wittmann-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] and which exhibited considerable differences in cross-linking efficiency in the GTP form as compared to the GDP form of the protein. The described affinity labeling technique of cross-linking [alpha-32P]GTP with GTP-binding proteins can be used as a general analytical method for the detection and identification of consensus elements in GTPases from different organisms.

Topics: Affinity Labels; Amino Acid Sequence; Binding Sites; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Molecular Sequence Data; Oxidation-Reduction; Periodic Acid; Protein Structure, Secondary; Proto-Oncogene Proteins p21(ras)