Analysis of the sorting signals directing NADH-cytochrome b5 reductase to two locations within yeast mitochondria.
Mitochondrial NADH-cytochrome b5 reductase (Mcr1p) is encoded by a single nuclear gene and imported into two different submitochondrial compartments: the outer membrane and the intermembrane space. We now show that the amino-terminal 47 amino acids suffice to target the Mcr1 protein to both destinations. The first 12 residues of this sequence function as a weak matrix-targeting signal; the remaining residues are mostly hydrophobic and serve as an intramitochondrial sorting signal for the outer membrane and the intermembrane space. A double point mutation within the hydrophobic region of the targeting sequence virtually abolishes the ability of the precursor to be inserted into the outer membrane but increases the efficiency of transport into the intermembrane space. Import of Mcr1p into the intermembrane space requires an electrochemical potential across the inner membrane, as well as ATP in the matrix, and is strongly impaired in mitochondria lacking Tom7p or Tim11p, two components of the translocation machineries in the outer and inner mitochondrial membranes, respectively. These results indicate that intramitochondrial sorting of the Mcr1 protein is mediated by specific interactions between the bipartite targeting sequence and components of both mitochondrial translocation systems.
Haucke
V
V
Biozentrum, Department of Biochemistry, University of Basel, Switzerland. volker.hauke@yale.edu
Ocana
C S
CS
Hönlinger
A
A
Tokatlidis
K
K
Pfanner
N
N
Schatz
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism; D002451:Cell Compartmentation; D003579:Cytochrome Reductases / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D005656:Fungal Proteins / Q000378:metabolism; D007425:Intracellular Membranes / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008928:Mitochondria / Q000201:enzymology*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D025261:Mitochondrial Proton-Translocating ATPases; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship
D002352:Carrier Proteins; D005656:Fungal Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; C099634:TOM7 protein, S cerevisiae; D000255:Adenosine Triphosphate; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D025261:Mitochondrial Proton-Translocating ATPases; C102550:TIM11 protein, S cerevisiae