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Translocation of proteins across mitochondrial membranes driven by a chaperone-based “import motor”

The vast majority of the hundreds of proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes. Thus, efficient import of proteins is critical for mitochondrial function. The import motor required for driving proteins across the inner membrane into the matrix is composed of 5 essential components, with the matrix Hsp70 molecular chaperone, at its core. Our goal is to dissect the specialized regulated protein:protein interactions amongst the components and the translocon channel that have evolved to drive efficient translocation of polypeptides across the membrane. 

Mitochondrial Hsp70 is tethered to the import channel via interaction with the peripheral membrane protein Tim44. The J-protein partner of mtHsp70, Pam18, is also tethered to the translocon, in part via interaction with Pam16. Having purified proteins in hand, we are now able to analyze interactions amongst these 4 proteins, and to ask how regulation of their interactions serves to drive protein import. Our goal is to develop a comprehensive model for import of proteins into the mitochondrial matrix that is consistent with genetic and in organellar mitochondrial import studies, as well as biochemical analysis of mutant proteins. 

This mitochondrial import system also serves as a “case study” of how an “all-purpose” Hsp70 that has maintained its involvement in general protein folding has evolved specialized, tightly regulated protein:protein interactions that allows it to carry a specialized function, protein translocation.

Questions we are addressing include:

  • Regulation of the Tim44:Ssc1 interaction. What is the molecular basis of the Tim44:Ssc1 interaction? What is the role of unfolded client protein, nucleotide and the nucleotide release factor in regulating the Tim44:Ssc1 interaction?
  • Tim44 as a scaffold for the entire import motor. We will test the hypothesis that Tim44 serves as a scaffold for all the import motor components, thus positioning the J-domain of Pam18 appropriately for efficient stimulation of Hsp70’s ATPase activity. Does Pam16 serve to tether Pam18 to the translocon via its interaction with Tim44 or to regulate Pam18’s ATPase stimulatory ability, or both?
  • Functional interaction of Tim44 with the import channel.  Tim17 and Tim23 compose the import channel. in organellar studies indicate a direct interaction between them and Tim44. We will analyze these interactions with a long-term goal of reconstituting the import apparatus of the inner membrane.

Selected Craig Lab Publications:

Schiller D, Cheng YC, Liu Q, Walter W, Craig EA. (2008) Residues of Tim44 involved in both association with the translocon of the inner mitochondrial membrane and regulation of mtHsp70 tethering.  Mol Cell Biol. 28:4424-33. [PDF]

D'Silva PR, Schilke B, Hayashi M, Craig EA. (2008) Interaction of the j-protein heterodimer pam18/pam16 of the mitochondrial import motor with the translocon of the inner membrane. Mol Biol Cell. 19:424-32. [PDF]

D'Silva PR, Schilke B, Walter W, Craig EA. (2005) Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane. Proc Natl Acad Sci U S A. 102:12419-24. [PDF]

D'Silva P, Liu Q, Walter W, Craig EA. (2004) Regulated interactions of mtHsp70 with Tim44 at the translocon in the mitochondrial inner membrane. Nat Struct Mol Biol. Nov;11(11):1084-91. [PDF]

D'Silva PD, Schilke B, Walter W, Andrew A, Craig EA. (2003) J protein cochaperone of the mitochondrial inner membrane required for protein import into the mitochondrial matrix. Proc Natl Acad Sci (USA).100(24):13839-44. [PDF]

Liu Q, D'Silva P, Walter W, Marszalek J, and Craig EA. (2003) Regulated cycling of mitochondrial Hsp70 at the protein import channel. Science. 300:139-41. [PDF]