The mitochondrial tricarboxylate carrier

Area of Science:

• Biochemistry
• Molecular Biology
• Mitochondrial Transport

Background:

• The tricarboxylate carrier is crucial for mitochondrial function, but its precise structure and genetic basis were previously uncharacterized.
• Previous purification efforts by different groups yielded proteins of varying sizes (30-38 kDa), suggesting potential challenges in isolation or differing protein forms.

Purpose of the Study:

• To purify and characterize the tricarboxylate carrier protein from rat liver mitochondria.
• To determine the protein sequence and genetic basis of the tricarboxylate carrier.
• To compare the tricarboxylate carrier's structure to other known mitochondrial carrier proteins.

Main Methods:

• Purification of the tricarboxylate carrier using silica gel 60 chromatography.
• Peptide mapping and partial sequencing of purified protein fragments.
• cDNA cloning via polymerase chain reaction and lambda ZAP library screening.
• Sequencing of the full-length cDNA and deduced amino acid sequence.

Main Results:

• A 37-38 kDa protein with specific citrate transport activity was purified.
• Peptide mapping indicated structural differences between independently purified proteins.
• A 2,986 bp cDNA clone was isolated, containing a 1071 bp open reading frame encoding a 322 amino acid protein (35.5 kDa).
• The tricarboxylate carrier sequence showed no significant homology to other mitochondrial carrier family members and lacked tripartite internal homology.
• mRNA expression was detected in rat liver and brain, but not in heart.

Conclusions:

• The tricarboxylate carrier is a distinct protein, structurally unique among mitochondrial carriers.
• The molecular and genetic basis of tricarboxylate transport has been elucidated.
• Tissue-specific expression suggests specialized roles in liver and brain metabolism.