Premium
Isolation and mass spectrometric analysis of native protein complexes in rat liver mitochondrial contact sites
Author(s) -
Lee Kwangwon,
Kerner Janos,
Hoppel Charles
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.988.8
Subject(s) - translocase , carnitine , biochemistry , chemistry , molecular mass , gene isoform , bacterial outer membrane , mitochondrion , adenine nucleotide translocator , inner mitochondrial membrane , nucleotide , microbiology and biotechnology , enzyme , biology , gene , escherichia coli , chromosomal translocation
Contact sites (CS) are structures where the mitochondrial inner and outer membrane are fused and can be isolated by discontinuous sucrose gradient centrifugation of crude outer membrane derived from swollen/shrunk rat liver mitochondria. We used a combination of Blue‐Native PAGE (BNE) and LC‐MS/MS to isolate and characterize the protein complexes present in CS. CS proteins were extracted with Triton X‐100 and separated into eleven different size bands by BNE. All eight bands, analyzed by LC‐MS/MS, contained voltage‐dependent anion channel (three isoforms), long‐chain acyl‐CoA synthetase 1, phosphate carrier. Four bands between 600 kDa and 960 kDa contained carnitine palmitoyltransferase 1a, very long‐chain acyl‐CoA dehydrogenase, and the trifunctional protein (both α and Î 2 ). In the same four bands, adenine nucleotide translocase (two isoforms) and apoptosis‐inducing factor 1 were identified. Of interest, carnitine‐acylcarnitine translocase was found only in the bands between 140 kDa and 400 kDa. Based on the proteins identified, liver mitochondrial CS from fed rats contain protein complexes of various high molecular weight. We propose these protein complexes of various sizes represent carnitine transport system as well as very long chain fatty acid oxidation enzymes, nucleotide transport system and apoptosis system. Supported by the NIH grant DK‐066107