Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes.
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.
D000818:Animals; D002352:Carrier Proteins / Q000032:analysis; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003911:Dextrans; D004229:Dithiothreitol; D018870:Endoplasmic Reticulum, Rough / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D008561:Membrane Fusion / Q000187:drug effects; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000378:metabolism*; D052066:N-Ethylmaleimide-Sensitive Proteins; D010504:Periodic Acid / Q000494:pharmacology; D011092:Polyethylene Glycols; D051381:Rats; D012710:Serum Albumin, Bovine / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D033921:Vesicular Transport Proteins; D015032:Zinc / Q000494:pharmacology
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D003911:Dextrans; D013438:Sulfhydryl Compounds; D013439:Sulfhydryl Reagents; D033921:Vesicular Transport Proteins; D010504:Periodic Acid; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D006160:Guanosine Triphosphate; C009288:metaperiodate; D052066:N-Ethylmaleimide-Sensitive Proteins; C498737:Nsf protein, rat; D008274:Magnesium; D015032:Zinc; D005033:Ethylmaleimide; D004229:Dithiothreitol