Studies on the sedimentation behavior of the Neurospora crassa plasma membrane H(+)-ATPase synthesized in vitro and integrated into homologous microsomal membranes.
RNA transcripts that encoded the Neurospora crassa plasma membrane H(+)-ATPase (pma+), a polytopic integral membrane protein, and the pma+344, a truncated pma+ with the amino terminal 344 amino acids, were translated in a N. crassa in vitro system. The microsomal membranes integrated products were insensitive to extraction by Na2CO3 (pH 11.5). The velocity sedimentation behavior of the in vitro synthesized pma+ were examined under various conditions. The pma+ migrated on linear sucrose gradients as aggregates which were heterogeneous in size, in the regions of 9-13 S; whereas, these values were reduced when Triton X-100 was presence in the gradients. The formation of these aggregates is interpreted to suggest a mechanism that maintains this polytopic integral membrane protein in a soluble form until it is targeted to the membranes. The sedimentation coefficient of the Triton X-100 solubilized microsomal membranes integrated pma+ corresponded roughly to a monomer of the pma+. Furthermore, a comparison of the trypsin cleavage patterns of the in vitro synthesized pma+ and of the microsomal membranes integrated pma+ suggest that they have different tertiary, or quaternary, structures. The latter did not give the characteristic trypsin cleavage patterns that have been observed for the native pma+ in the presence of its ligands MgATP and vanadate (Addison, R. and Scarborough, G.A. (1982) J. Biol. Chem. 257, 10421-10426). This was interpreted to suggest that the microsomal membranes integrated pma+ cannot interact with its substrate, suggesting that it is catalytically inactive.
Addison
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Department of Biochemistry, University of Tennessee, Health Science Center, Memphis 38163.
D002462:Cell Membrane / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology*; D008970:Molecular Weight; D009492:Neurospora crassa / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010957:Plasmids; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D014158:Transcription, Genetic; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D012331:RNA, Fungal; D004247:DNA; D014357:Trypsin; D006180:Proton-Translocating ATPases