title: Role of Pam16's degenerate J domain in protein import across the mitochondrial inner membrane.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: D'Silva
authors.forename: Brenda
authors.initials: B
authors.lastname: Schilke
authors.forename: William
authors.initials: W
authors.lastname: Walter
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Craig
mesh_terms: 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
chemical_list: 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