Abstract
D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria. It spans the membrane once in an Nin-Cout orientation with the bulk of the protein residing as a folded domain in the intermembrane space. D-LD is synthesized as a precursor with an N-terminal cleavable presequence and is imported into the mitochondria in a Deltapsi-dependent, but mt-Hsp70-independent manner. Upon import in vitro D-LD folds in the intermembrane space to attain a conformation indistinguishable from endogenous D-LD. Sorting of D-LD to the inner membrane is directed by a composite topogenic signal consisting of the hydrophobic transmembrane segment and a cluster of charged amino acids C-terminal to it. We propose a model for the mode of operation of the sorting signal of D-LD. This model also accounts for the driving force of translocation across the outer membrane, in the apparent absence of mt-Hsp70-dependent assisted import and involves the folding of the D-LD in the intermembrane space.
Highlights
D-Lactate dehydrogenase (D-LD) is located in the inner membrane of mitochondria
How do they act to ensure the sorting of proteins to the inner membrane? Recently we have described that the topogenic signals of a subset of inner membrane proteins serve as export signals directing the export of domains of the protein from the matrix to the intermembrane space following the complete import of the preprotein into the matrix [1,2,3]
To determine the submitochondrial localization of the D-LD, isolated mitochondria were subjected to protease treatment and subfractionation by hypotonic swelling, which disrupts the integrity of the outer membrane while leaving the inner membrane intact (Fig. 1A)
Summary
(Received for publication, December 8, 1997, and in revised form, January 13, 1998) From the Institut fur Physiologische Chemie, Geethestrasse 33, 80336 Munchen, Germany and §Centre de Genetique Moleculaire CNRS, Universite Pierre et Marie Curie, 91190 Gif-sur-Yvette, France. Nuclear encoded proteins of the inner membrane of mitochondria contain topogenic signals which function to sort them to the membrane following their import These topogenic signals comprise hydrophobic cores of varying length and are flanked usually by charged amino acids. In a small number of cases they are proteolytically cleaved following the sorting event These topogenic signals are distinct from mitochondrial targeting sequences that serve to target the precursor initially to the mitochondria and to initiate membrane potential (⌬)-dependent translocation across the inner membrane. In some cases these topogenic signals are located after N-terminal mitochondrial targeting signals (presequences) and operate in conjunction with them. We present here information on the topogenic signal sequence and energetic requirements necessary for D-LD to achieve this membrane orientation
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