Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
41
pubmed:dateCreated
2000-11-13
pubmed:abstractText
The topography of rat glycerophosphate acyltransferase (GAT) in the transverse plane of the mitochondrial outer membrane (MOM) was investigated. Computer analysis of the amino acid (aa) sequence derived from rat mitochondrial GAT cDNA (GenBanktrade mark accession nos. and ) predicts the presence of two possible transmembrane domains (aa 473-493 and 574-594) separated by an 80-aa stretch (aa 494-573). To determine the actual orientation of the native protein, we prepared anti-peptide antibodies to three regions: one in between (aa 543-559) and the other two (aa 420-435 and 726-740) flanking the two putative transmembrane regions. Both immunoreaction and immunoprecipitation experiments employing intact and solubilized mitochondria indicate that regions on the N- and C-terminal sides of the transmembrane regions are sequestered on the inner surface of the MOM, while the region between the transmembrane domains is present on the cytosolic face of the MOM. Additionally, two green fluorescent protein (GFP) fusion proteins consisting of full-length GAT fused to GFP at either the C terminus or inserted 115 amino acids from the N terminus were also constructed to determine the orientation of the N and C termini. COS-1 cells expressing these fusion proteins were fractionated to obtain mitochondria. Protease digestion of intact and solubilized COS-1 cell mitochondria revealed that the GFP domains of these fusion proteins are sequestered on the inner side of the MOM. The present findings indicate that GAT is a dual-spanning, transmembrane protein adopting an inverted "U" conformation in the transverse plane of the MOM, where the N and C termini are sequestered on the inner surface of the MOM, while aa 494-573 are exposed on the cytosolic surface of the MOM.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
13
pubmed:volume
275
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
31668-73
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed-meshheading:10924502-Animals, pubmed-meshheading:10924502-Antibodies, pubmed-meshheading:10924502-COS Cells, pubmed-meshheading:10924502-Computer Simulation, pubmed-meshheading:10924502-Cytosol, pubmed-meshheading:10924502-Glycerol-3-Phosphate O-Acyltransferase, pubmed-meshheading:10924502-Intracellular Membranes, pubmed-meshheading:10924502-Male, pubmed-meshheading:10924502-Membrane Proteins, pubmed-meshheading:10924502-Mitochondria, Liver, pubmed-meshheading:10924502-Precipitin Tests, pubmed-meshheading:10924502-Protein Footprinting, pubmed-meshheading:10924502-Protein Structure, Tertiary, pubmed-meshheading:10924502-Rats, pubmed-meshheading:10924502-Rats, Sprague-Dawley, pubmed-meshheading:10924502-Recombinant Fusion Proteins, pubmed-meshheading:10924502-Solubility, pubmed-meshheading:10924502-Transfection, pubmed-meshheading:10924502-Trypsin
pubmed:year
2000
pubmed:articleTitle
Identification of two transmembrane regions and a cytosolic domain of rat mitochondrial glycerophosphate acyltransferase.
pubmed:affiliation
Department of Biological Sciences, St. John's University, Jamaica, New York 11432, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.