Domain structure and lipid interaction of recombinant yeast Tim44.
Tim44 is an essential component of the machinery that mediates the translocation of nuclear-encoded proteins across the mitochondrial inner membrane. It functions as a membrane anchor for the ATP-driven protein import motor whose other subunits are the mitochondrial 70-kDa heat-shock protein (mhsp70) and its nucleotide exchange factor, mGrpE. To understand how this motor is anchored to the inner membrane, we have overexpressed Tim44 in Escherichia coli and studied the properties of the pure protein and its interaction with model lipid membranes. Limited proteolysis and analytical ultracentrifugation indicate that Tim44 is an elongated monomer with a stably folded C-terminal domain. The protein binds strongly to liposomes composed of phosphatidylcholine and cardiolipin but only weakly to liposomes containing phosphatidylcholine alone. Studies with phospholipid monolayers suggest that Tim44 binds to phospholipids of the mitochondrial inner membrane both by electrostatic interactions and by penetrating the polar head group region.
Weiss
C
C
Department of Biochemistry, Tel-Aviv University, Tel-Aviv 69978, Israel.
Oppliger
W
W
Vergères
G
G
Demel
R
R
Jenö
P
P
Horst
M
M
de Kruijff
B
B
Schatz
G
G
Azem
A
A
D002308:Cardiolipins / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli; D007700:Kinetics; D008081:Liposomes / Q000378:metabolism*; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D016366:Open Reading Frames; D010713:Phosphatidylcholines / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013395:Sucrose / Q000378:metabolism
D002308:Cardiolipins; D002352:Carrier Proteins; D008081:Liposomes; D046911:Macromolecular Substances; D008563:Membrane Lipids; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C077016:TIM44 protein, S cerevisiae; D013395:Sucrose; C017251:1,2-oleoylphosphatidylcholine