Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane.
Translocation of proteins across the mitochondrial inner membrane is an essential process requiring an import motor having mitochondrial Hsp70 (mtHsp70) at its core. The J protein partner of mtHsp70, Pam18, is an integral part of this motor, serving to stimulate the ATPase activity of mtHsp70. Pam16, an essential protein having an inactive J domain that is unable to stimulate mtHsp70's ATPase activity, forms a heterodimer with Pam18, but its function is unknown. We set out to test the importance of three properties of Pam16: (i) a stable interaction between Pam16 and Pam18, (ii) the inability of Pam16's degenerate J domain to stimulate Ssc1's ATPase domain, and (iii) the innately lower stimulatory activity of the Pam16:Pam18 heterodimer, compared to Pam18 alone. Neither substantial reduction in the ability of Pam18 to stimulate Ssc1's ATPase activity, nor the presence of an active J domain in Pam16, had deleterious effects on cell growth, indicating the lack of importance of two of these biochemical properties. However, a stable interaction between Pam16's degenerate J domain and Pam18's J domain was found to be critical for function. Alterations that destabilized the Pam16:Pam18 heterodimer had deleterious effects on cell growth and mitochondrial protein import; intragenic suppressors that restored robust growth also restored heterodimer stability. Our results support the idea that Pam16's J-like domain strongly interacts with Pam18's J domain, leading to a productive interaction of Pam18 with mtHsp70 at the import channel.
D'Silva
Patrick R
PR
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Schilke
Brenda
B
Walter
William
W
Craig
Elizabeth A
EA
D001693:Biological Transport, Active; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D007425:Intracellular Membranes / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D020409:Molecular Motor Proteins; C480348:PAM18 J protein, S cerevisiae; C484526:Pam16 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; C101767:SSC1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000252:Calcium-Transporting ATPases