Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling

Abstract

To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [³H]diazofluorene ([³H]DAF) and 1-azidopyrene (1-AP) to tag the membrane proteins which were then identified by amino-terminal sequence analysis of labeled fragments isolated from proteolytic digests by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by reverse-phase high-performance liquid chromatography. In addition to AChR subunits, identified polypeptides include the 95 kDa α-subunit of the (Na⁺+K⁺)-ATPase, the 89 kDa voltage-gated chloride channel (CLC-0), the 105 kDa SITS-binding protein, and 32 and 34 kDa polypeptides identified as Torpedo homologues of the mitochondrial membrane ATP/ADP carrier protein and the voltage-dependent anion channel (VDAC), respectively. Further, individual amino acids that reacted with [³H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the α-subunit of the (Na⁺+K⁺)-ATPase and in a putative transmembrane β-strand in VDAC. Collectively these results demonstrate that [³H]DAF/1-AP photolabeling provides an effective method for tagging the membrane-associated segments of polypeptides in a way that makes it easy to isolate the labeled polypeptide or polypeptide fragments by fluorescence and then to identify amino acids at the lipid-protein interface by ³H release.