Trichomonas vaginalis Hmp35, a Putative Pore-forming Hydrogenosomal Membrane Protein, Can Form a Complex in Yeast Mitochondria

Sabrina D Dyall,Dianna C Lester,Rachel E Schneider,Maria G Delgadillo-Correa,Evelyn Plümper,Ariadna Martinez,Carla M Koehler,Patricia J Johnson

doi:10.1074/jbc.m304032200

Abstract

An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis. This protein has no known homologue and exists as a stable 300-kDa complex, termed HMP35, in membranes of the hydrogenosome. By using blue native gel electrophoresis, we found the HMP35 complex to be stable in 2 m NaCl and up to 5 m urea. The endogenous Hmp35 protein was largely protease-resistant. The protein has a predominantly beta-sheet structure and predicted transmembrane domains that may form a pore. Interestingly, the protein has a high number of cysteine residues, some of which are arranged in motifs that resemble the RING finger, suggesting that they could be coordinating zinc or another divalent cation. Our data show that Hmp35 forms one intramolecular but no intermolecular disulfide bonds. We have isolated the HMP35 complex by expressing a His-tagged Hmp35 protein in vivo followed by purification with nickel-agarose beads. The purified 300-kDa complex consists of mostly Hmp35 with lesser amounts of 12-, 25-27-, and 32-kDa proteins. The stoichiometry of proteins in the complex indicates that Hmp35 exists as an oligomer. Hmp35 can be targeted heterologously into yeast mitochondria, despite the lack of homology with any yeast protein, demonstrating the compatibility of mitochondrial and hydrogenosomal protein translocation machineries.

Highlights

An abundant integral membrane protein, Hmp35, has been isolated from hydrogenosomes of Trichomonas vaginalis
Isolation and Characterization of Hmp35, a Hydrogenosomal Membrane Protein—To pursue the identification of hydrogenosomal membrane proteins from T. vaginalis, we targeted a number of proteins in the 35– 40-kDa range (Fig. 1A, lane 3, MP40) that are of intermediate abundance
We demonstrate the application of blue native gel electrophoresis for hydrogenosomal complex separation and show that the Hmp35 protein is part of an integral membrane complex of about 300 kDa, HMP35, which is resistant to high concentrations of protease, urea, and salt

Summary

The abbreviations used are

Heat shock protein; MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TLCK, N␣-p-tosyl-L-lysine chloromethyl ketone; DTT, dithiothreitol; TCEP, Tris[2-carboxyethyl] phosphine hydrochloride; AMS, 4-acetamide-4-maleimidylstilbene-2,2-disulfonic acid; PMSF, phenylmethylsulfonyl fluoride; BN-PAGE, blue native polyacrylamide gel electrophoresis; IMS, intermembrane space. We describe a novel hydrogenosomal membrane protein, Hmp, that has no known homologue in prokaryotes or eukaryotes to date. It is a relatively abundant membrane protein that exists as an integral membrane complex. It has no primary sequence homology to those proteins, Hmp has a predominant predicted ␤-sheet structure similar to that found in the outer membrane proteins of Gram-negative bacteria, mitochondria, and plastids [22]. Taken together, these data suggest a similar function for Hmp. We demonstrate that despite the absence of a homologue in yeast or of a recognizable mitochondrial targeting sequence, the Hmp can be heterologously expressed in yeast and is targeted to yeast mitochondrial membranes in vivo

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION