Saccharomyces cerevisiae Porin Pore Forms Complexes with Mitochondrial Outer Membrane Proteins Om14p and Om45p

Susann Lauffer,Katrin Mäbert,Cornelia Czupalla,Theresia Pursche,Bernard Hoflack,Gerhard Rödel,Udo Krause-Buchholz

doi:10.1074/jbc.m111.328328

Abstract

Numerous transport processes occur between the two mitochondrial (mt) membranes due to the diverse functions and metabolic processes of the mt organelle. The metabolite and ion transport through the mt outer membrane (OM) is widely assumed to be mediated by the porin pore, whereas in the mt inner membrane (IM) specific carriers are responsible for transport processes. Here, we provide evidence by means of Blue Native (BN)-PAGE analysis, co-immunoprecipitation, and tandem affinity purification that the two mt OM proteins Om14p and Om45p associate with the porin pore. Porin molecules seem to assemble independently to build the core unit. A subpopulation of these core units interacts with Om14p and Om45p. With preparative tandem affinity purification followed by MS analysis, we could identify interaction partners of this OM complex, which are mainly localized within the mt IM and function as carriers for diverse molecules. We propose a model for the role of the two OM proteins in addressing the porin pore to bind to specific channels in the mt IM to facilitate transport of metabolites.

Highlights

No information is available about the association of the mitochondrial porin pore with the major outer membrane proteins Om14p and Om45p
Om14p, Om45p, and Por1p Interact with Each Other—The co-localization and similar steady state levels of Por1p, Om14p, and Om45p in the mt outer membrane (OM) [11,12,13] led us to speculate that these proteins might interact
The mt OM proteome of S. cerevisiae is dominated by the presence of three transmembrane proteins Por1p, Om14p, and Om45p [11,12,13]

Summary

Background

No information is available about the association of the mitochondrial porin pore with the major outer membrane proteins Om14p and Om45p. The transport of metabolites and ions through the mt OM is widely assumed to occur unspecifically via the porin pore (Por1p in Saccharomyces cerevisiae). In mammalian organisms, this pore is called the voltage-dependent anion channel (VDAC) as it selects for negatively charged molecules like ATP Benzenesulfonyl fluoride hydrochloride; BN, Blue Native; DIGE, difference in gel electrophoresis; IM, inner membrane; IMS, inner membrane space; MCF, mitochondrial carrier family; OM, outer membrane; PI-Mix, proteinase inhibitor mixture; TAP, tandem affinity purification; TEV, tobacco etch virus; TSPO, translocator protein of the mt OM; VDAC, voltage-dependent anion channel. Our data are summarized in an interaction model suggesting the involvement of Om14p and Om45p in addressing the porin pore to bind to specific channels in the mt IM

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION