Abstract
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.
Highlights
The mitochondrial outer membrane (MOM)1 is the demarcatory barrier between the mitochondrial interior and cytosol
The present findings indicate that glycerophosphate acyltransferase (GAT) is a dualspanning, 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
Analysis of rat mitochondrial GAT resulted in six candidate hydrophobic regions (Fig. 1A, boxes 1– 6), wherein two of those regions, aa 473– 493 and 574 –594, elicited a rating of “certain” transmembrane regions (Fig. 1A, boxes 3 and 5)
Summary
The mitochondrial outer membrane (MOM)1 is the demarcatory barrier between the mitochondrial interior and cytosol. 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.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: 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.
