Identification and characterization of membrane-associated polypeptides in Torpedo nicotinic acetylcholine receptor-rich membranes by hydrophobic photolabeling.
To identify membrane-associated polypeptides present in Torpedo nicotinic acetylcholine receptor (AChR)-rich membranes, we used hydrophobic photolabeling with [(3)H]diazofluorene ([(3)H]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 alpha-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 [(3)H]DAF and therefore likely to be in contact with lipid were identified in the transmembrane segment M3 of the alpha-subunit of the (Na(+)+K(+))-ATPase and in a putative transmembrane beta-strand in VDAC. Collectively these results demonstrate that [(3)H]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 (3)H release.
Blanton
M P
MP
Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA.
Lala
A K
AK
Cohen
J B
JB
D000595:Amino Acid Sequence; D000818:Animals; D017302:Annexins / Q000737:chemistry; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry*; D003979:Diazonium Compounds; D004591:Electrophoresis, Polyacrylamide Gel; D005449:Fluorenes; D005456:Fluorescent Dyes; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D019920:Photoaffinity Labels; D018272:Porins / Q000737:chemistry; D021122:Protein Subunits; D011721:Pyrenes; D051381:Rats; D011978:Receptors, Nicotinic / Q000032:analysis* / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000302:isolation & purification; D014101:Torpedo; D014316:Tritium; D050994:Voltage-Dependent Anion Channels
D017302:Annexins; D003979:Diazonium Compounds; D005449:Fluorenes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010446:Peptide Fragments; D019920:Photoaffinity Labels; D018272:Porins; D021122:Protein Subunits; D011721:Pyrenes; D011978:Receptors, Nicotinic; D050994:Voltage-Dependent Anion Channels; D014316:Tritium; C028591:azidopyrene; C052770:2-diazofluorene; D000254:Sodium-Potassium-Exchanging ATPase