Abstract
Reconstituted proteoliposomes containing functional Neurospora crassa plasma membrane H(+)-ATPase molecules oriented predominantly with their cytoplasmic surface exposed were treated with trypsin and then subjected to Sepharose CL-6B column chromatography to remove the liberated peptides. The peptides remaining associated with the liposomes were then separated from the phospholipid by Sephadex LH-60 column chromatography and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Six H(+)-ATPase peptides with approximate molecular masses of 7, 7.5, 8, 10, 14, and 21 kDa were found to be tightly associated with the liposomal membrane. Amino acid sequencing of the 7-, 7.5-, and 21-kDa peptides in the LH-60 eluate identified them as H(+)-ATPase fragments beginning at residues 99 or 100, 272, and 660, respectively. After further purification, the approximately 10- and 14-kDa peptides were also similarly identified as beginning at residues 272 and 660. The approximately 8-kDa fragment was purified further but could not be sequenced, presumably indicating NH2-terminal blockage. To identify which of the liposome-associated peptides are embedded in the membrane, H(+)-ATPase molecules in the proteoliposomes were labeled from the hydrophobic membrane interior with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and cleaved with trypsin, after which the membrane-associated peptides were purified and assessed for the presence of label. The results indicate that the approximately 7-, 7.5-, and 21-kDa peptides are in contact with the lipid bilayer whereas the approximately 8-kDa peptide is not. Taken together with the results of our recent analyses of the peptides released from the proteoliposomes, this information establishes the transmembrane topography of nearly all of the 919 residues in the H(+)-ATPase molecule.
Highlights
Neurospora cra88a plasma membrane H+-ATPasemol- reported direct protein chemical analyses of the transmemecules oriented predominantly with their cytoplasmic brane topography of the H'-ATPase molecule ( 1, 2 )
The peptides remaining associated with theliposomeswere separated fromthe phospholipid by Sephadex LH-60 column chromatography and analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis
The peptides released by this treatment were removed by chromatography of the mixture on a column of Sephadex CL-GB, resulting in liposomes containing only
Summary
Neurospora cra88a plasma membrane H+-ATPasemol- reported direct protein chemical analyses of the transmemecules oriented predominantly with their cytoplasmic brane topography of the H'-ATPase molecule ( 1 , 2 ). In these surface exposed were treated with trypsin and studies, reconstituted proteoliposomes demonstrated to consubjected to Sepharose CL-GB column chromatography tain H+-ATPasemolecules oriented predominantly with their to remove the liberatedpeptides. Amino acid normally cytoplasmic surface facing outward werecleaved with trypsin,and the peptides released were purified and identified by NHz-terminal amino acid sequence analyses The results of these experiments identified 417 residues of the H'-ATPase molecule as located on the cytoplasmic surface of the membrane, including the NHz anCd OOH termini. Taken together with the results of our recent step gradient centrifugation were carried out as described by Hennesanalyses of the peptides released from the proteolipo- sey and Scarborough [1].The specific ATPase activities in the pressomes, this information establishes the transmembrane topography of nearly allof the 919 residues in theH+ATPase molecule
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.