Characterization and Developmentally Regulated Localization of the Mitochondrial Carrier Protein Homologue MCP6 from Trypanosoma brucei

Abstract

The order Kinetoplastida includes among its members a number of important human and veterinary pathogens, such as the African trypanosome Trypanosoma brucei (83). This parasite has a complex life cycle that alternates between the bloodstream form found in the blood and tissue fluids of mammals and the procyclic (insect) form in the midgut of the tsetse fly (46, 83). T. brucei undergoes a series of metabolic and morphological changes in order to adapt to these distinct host environments (45, 46). Bloodstream-form T. brucei metabolizes glucose to pyruvate and glycerol, obtaining ATP by substrate-level phosphorylation during glycolysis (48, 55). The mitochondria of this life form are highly reduced and lack key enzymes and components of the tricarboxylic acid cycle; their role in energy metabolism is probably restricted to the reoxidation of glycerol-3 phosphate by the mitochondrial alternative oxidase (20,48). This is in contrast to the procyclic form, which metabolizes amino acids and has a well-developed mitochondrion in which ATP is generated by a combination of substratelevel and oxidative phosphorylation (16,48,73,82).In both life forms of T. brucei, most of the glycolytic enzymes are found within a peroxisome-like organelle called the glycosome (14,48,60,61). This unique compartmentation of glycolytic enzymes has been shown to be essential for trypanosome survival (5,11,22,25,51). It has been proposed that the glycosomal membrane forms a barrier to ATP and ADP, insulating the enzymes of the glycosomal matrix from the ADP/ ATP ratio in the cytosol (5).So far, no glycosomal or mitochondrial metabolite carriers have been identified for T. brucei, although they most probably play a key role in the regulation of energy metabolism. The characterization of these carriers should make it possible to build more-accurate biochemical and mathematical models of trypanosome energy metabolism (5).The mitochondrial carrier family (MCF) was initially defined as a group of proteins that are located in the inner mitochondrial membrane and mediate the transport of a large range of metabolic intermediates (3,32,35,(57)(58)(59)86). Recently, several structurally and functionally related carrier proteins were also found in the membranes of peroxisomes (52,81,88,90). We therefore wondered whether such proteins might also be found in kinetoplastid glycosomes. The recently completed genome sequence of T. brucei (8) contains about 29 genes encoding different proteins of the mitochondrial carrier family. In this paper, we describe the characterization of MCP6, a novel mitochondrial carrier protein homologue from T. brucei. MATERIALS AND METHODSCulture and transfection. Trypanosoma brucei cell lines were cultured in either HMI-9 medium for bloodstream cell lines (29) or MEM-PROS medium for procyclic cell lines (56), supplemented with 10% (vol/vol) fetal calf serum (Sigma-Aldrich). Cells were transfected as described previously (9). In all experiments, "wild type" refers to bloodstream or procyclic T. brucei strain

The ADP/ATP Carrier and Its Relationship to Oxidative Phosphorylation in Ancestral Protist Trypanosoma brucei

“…An obvious candidate for such an activity would be a homolog of the ATP-Mg/P i transporter, a protein that is responsible for the electroneutral exchange between ATP-Mg 2Ϫ and HPO 4 2Ϫ , which can supplement the mt ATP pool in cells using the hydrolytic activity of F o F 1 -ATPase to maintain the ⌬m (68). However, a putative T. brucei homolog of the ATP-Mg/P i transporter (Tb927.4.1660, MCP6) was shown to be inactive for ATP and ADP transport (25,69). Thus, besides TbAAC, any additional carrier(s) responsible for ATP influx into the mitochondria of this excavate protist remains to be elucidated.…”

Functional Characterization of TbMCP5, a Conserved and Essential ADP/ATP Carrier Present in the Mitochondrion of the Human Pathogen Trypanosoma brucei

“…Immunodetection was performed using horseradish peroxidase (HRP)-coupled protein A (Bio-Rad) or HRP-coupled Myc antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and the enhanced chemiluminescence (ECL) kit from GE Healthcare. Immunofluorescence microscopy using paraformaldehyde-fixed trypanosomes was performed as described previously (17,33). 4Ј,6-Diamidino-2-phenyl-indole (DAPI) was used for the specific staining of nuclear and kinetoplastid (mitochondrial) DNA, the mitochondrial marker MitoTracker (Invitrogen) was used for the specific labeling of T. brucei mitochondria (34), and the TbMCP5 antibody was used for the specific labeling of TbMCP5.…”

Adenine nucleotide transporters in organelles: novel genes and functions

“…MCP6 is a mitochondrial carrier of 385 amino acids [48], and remarkably, it lacks an N-terminal extension with EF-hand calcium-binding motifs, and thus, transport is probably Ca 2? -independent.…”