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A dual role for mitochondrial heat shock protein 70 in membrane translocation of preproteins
B D Gambill1, W Voos, P J Kang
1Department of Biomolecular Chemistry, University of Wisconsin-Madison 53706.
Abstract:
The role of mitochondrial 70-kD heat shock protein (mt-hsp70) in protein translocation across both the outer and inner mitochondrial membranes was studied using two temperature-sensitive yeast mutants. The degree of polypeptide translocation into the matrix of mutant mitochondria was analyzed using a matrix-targeted preprotein that was cleaved twice by the processing peptidase. A short amino-terminal segment of the preprotein (40-60 amino acids) was driven into the matrix by the membrane potential, independent of hsp70 function, allowing a single cleavage of the presequence. Artificial unfolding of the preprotein allowed complete translocation into the matrix in the case where mutant mt-hsp70 had detectable binding activity. However, in the mutant mitochondria in which binding to mt-hsp70 could not be detected the mature part of the preprotein was only translocated to the intermembrane space. We propose that mt-hsp70 fulfills a dual role in membrane translocation of preproteins. (a) Mt-hsp70 facilitates unfolding of the polypeptide chain for translocation across the mitochondrial membranes. (b) Binding of mt-hsp70 to the polypeptide chain is essential for driving the completion of transport of a matrix-targeted preprotein across the inner membrane. This second role is independent of the folding state of the preprotein, thus identifying mt-hsp70 as a genuine component of the inner membrane translocation machinery. Furthermore we determined the sites of the mutations and show that both a functional ATPase domain and ATP are needed for mt-hsp70 to bind to the polypeptide chain and drive its translocation into the matrix.
Insights
Mitochondrial 70-kD heat shock protein (mt-hsp70) aids protein import into mitochondria by unfolding polypeptides and driving their transport across membranes. Its binding activity is crucial for completing translocation into the matrix.
Area of Science:
- Mitochondrial biology
- Protein translocation
- Molecular chaperones
Background:
- Mitochondrial protein import is essential for cellular function.
- Mitochondrial 70-kD heat shock protein (mt-hsp70) is implicated in protein translocation.
- The precise roles of mt-hsp70 in traversing mitochondrial membranes are not fully understood.
Purpose of the Study:
- To elucidate the specific functions of mt-hsp70 in protein translocation across mitochondrial membranes.
- To investigate the dual role of mt-hsp70 in polypeptide unfolding and matrix-targeted protein transport.
- To identify the molecular requirements for mt-hsp70 activity in protein import.
Main Methods:
- Utilized temperature-sensitive yeast mutants defective in mt-hsp70.
- Analyzed translocation of a matrix-targeted preprotein using processing peptidase cleavage.
- Assessed preprotein translocation and mt-hsp70 binding activity under varying conditions, including artificial unfolding.
Main Results:
- A membrane potential drives initial translocation of a preprotein segment, independent of mt-hsp70.
- Functional mt-hsp70 with binding activity facilitates complete translocation of unfolded preproteins into the matrix.
- In mutants lacking mt-hsp70 binding, preproteins translocate only to the intermembrane space.
- mt-hsp70's ATPase domain and ATP are necessary for polypeptide binding and matrix translocation.
Conclusions:
- mt-hsp70 plays a dual role: facilitating polypeptide unfolding and driving completion of matrix protein transport.
- mt-hsp70 is a critical component of the inner mitochondrial membrane translocation machinery.
- ATP-dependent binding of mt-hsp70 to polypeptides is essential for efficient mitochondrial protein import.