Protein Import into Subsarcolemmal and Intermyofibrillar Skeletal Muscle Mitochondria: DIFFERENTIAL IMPORT REGULATION IN DISTINCT SUBCELLULAR REGIONS

Mark Takahashi,David A Hood

doi:10.1074/jbc.271.44.27285

Abstract

To date, no studies have described the import of proteins in mitochondria obtained from skeletal muscle. In this tissue, mitochondria consist of the functionally and biochemically distinct intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions, which are localized in specialized cellular compartments. This mitochondrial heterogeneity in muscle could be due, in part, to differential rates of protein import. To evaluate this possibility, the import of precursor malate dehydrogenase and ornithine carbamyltransferase proteins was investigated in isolated IMF and SS mitochondria in vitro. Import of these was 3-4-fold greater in IMF compared with SS mitochondria as a function of time. This could account for the higher malate dehydrogenase enzyme activity in IMF mitochondria. Divergent import rates in IMF and SS mitochondria likely result from a differential reliance on various components of the import pathway. SS mitochondria possess a greater content of the molecular chaperones hsp60 and Grp75, yet import is lower than in IMF mitochondria. On the other hand, adriamycin inhibition studies illustrated a greater reliance on acidic phospholipids (i.e. cardiolipin) for the import process in SS mitochondria. Matrix ATP levels were 3-fold higher in IMF mitochondria, but experiments in which ATP depletion was performed with atractyloside and oligomycin illustrated a dissociation between import rates and levels of ATP. In contrast, a close relationship was found between the rate of ATP production (i.e. mitochondrial respiration) and protein import. When respiratory rates in IMF and SS mitochondria were equalized, import rates in both subfractions were similar. These data indicate that 1) import rates are more closely related to the rate of ATP production than the steady state ATP level, 2) import into IMF and SS mitochondrial subfractions is regulated differently, and 3) mitochondrial heterogeneity within a cell type can be due to differences in the rates of protein import, suggesting that this step is a potentially regulatable event in determining the final mitochondrial phenotype.

Highlights

To date, no studies have described the import of proteins in mitochondria obtained from skeletal muscle
Studies of skeletal muscle subject to conditions of chronic use or disuse have indicated that SS mitochondria are synthesized or degraded in more dramatic fashion than IMF mitochondria [20, 55,56,57]
This heterogeneous response of the two mitochondrial subfractions implies that a different pattern or regulation of mitochondrial biogenesis exists in distinct regions of the muscle cell, perhaps influenced by surrounding nuclear domains [58] or the proximity to environmental factors

Summary

Introduction

No studies have described the import of proteins in mitochondria obtained from skeletal muscle In this tissue, mitochondria consist of the functionally and biochemically distinct intermyofibrillar (IMF) and subsarcolemmal (SS) subfractions, which are localized in specialized cellular compartments. An approximate 3-fold greater rate of ATP synthesis occurs in IMF, compared with SS mitochondria [19] This may be an important determinant of protein import and could account for some of the observed differences in protein composition between these two subfractions. The primary purpose of the present study was to establish the measurement of protein import into skeletal muscle IMF and SS mitochondrial subfractions and to examine mechanisms of import related to the abundance of intramitochondrial chaperones, the ATP dependence of import, and the potential role of cardiolipin in the translocation process

Objectives

Methods

Results

Conclusion