Yeast prions, such as [ PSI ⁺ ], [ RNQ ⁺ ], and [ URE3 ], are heritable elements formed by proteins capable of acquiring self-perpetuating conformations. Their propagation is dependent on fragmentation of the amyloid protein complexes formed to generate the additional seeds necessary for conversion of nascent soluble protein to the prion conformation. We report that, in addition to its known role in [ RNQ ⁺ ] propagation, Sis1, a J-protein cochaperone of Hsp70 Ssa, is also specifically required for propagation of [ PSI ⁺ ] and [ URE3 ]. Whereas both [ RNQ ⁺ ] and [ URE3 ] are cured rapidly upon SIS1 repression, [ PSI ⁺ ] loss is markedly slower. This disparity cannot be explained simply by differences in seed number, as [ RNQ ⁺ ] and [ PSI ⁺ ] are lost with similar kinetics upon inhibition of Hsp104, a remodeling protein required for propagation of all yeast prions. Rather, in the case of [ PSI ⁺ ], our results are consistent with the partial impairment, rather than the complete abolition, of fragmentation of prion complexes upon Sis1 depletion. We suggest that a common set of molecular chaperones, the J-protein Sis1, the Hsp70 Ssa, and the AAA+ ATPase Hsp104, act sequentially in the fragmentation of all yeast prions, but that the threshold of Sis1 activity required for each prion varies.

The major Hsp70 of the mitochondrial matrix (Ssc1 in yeast) is critically important for the translocation of proteins from the cytosol, across the mitochondrial inner membrane, and into the matrix. Tim44, a peripheral inner membrane protein with limited sequence similarity to the J domain of J-type cochaperones, tethers Ssc1 to the import channel. Here we report that, unlike a J protein, Tim44 does not stimulate the ATPase activity of Ssc1, nor does it affect the stimulation by either a known mitochondrial J protein or a peptide substrate. Thus, we conclude that Tim44 does not function as a J protein cochaperone of Ssc1; rather, it tethers Ssc1 to the import channel through interactions independent of those critical for J protein function. However, a previously unstudied essential gene, PAM18 , encodes an 18-kDa protein that contains a J domain and is localized to the mitochondrial inner membrane. Pam18 stimulates the ATPase activity of Ssc1; depletion of Pam18 in vivo disrupts import of proteins into the mitochondrial matrix. We propose that Pam18 is the J protein partner for Ssc1 at the import channel and is critical for Ssc1's function in protein import.

Efficient movement of proteins across membranes is required for cell health. The translocation process is particularly challenging when the channel in the membrane through which proteins must pass is narrow—such as those in the membranes of the endoplasmic reticulum and mitochondria. Hsp70 molecular chaperones play roles on both sides of these membranes, ensuring efficient translocation of proteins synthesized on cytosolic ribosomes into the interior of these organelles. The “import motor” in the mitochondrial matrix, which is essential for driving the movement of proteins across the mitochondrial inner membrane, is arguably the most complex Hsp70-based system in the cell.

J proteins are obligate cochaperones of Hsp70s, stimulating their ATPase activity and thus allowing them to function in multiple cellular processes. In most cellular compartments, an Hsp70 works with multiple, structurally divergent J proteins. To better understand the functional specificity of J proteins and the complexity of the Hsp70:J protein network, we undertook a comprehensive analysis of 13 J proteins of the cytosol of the yeast Saccharomyces cerevisiae . Phenotypes caused by the absence of four proteins, Sis1, Jjj1, Jjj3, and Cwc23, could not be rescued by overexpression of any other cytosolic J protein, demonstrating the distinctive nature of J proteins. In one case, that of Zuo1, the phenotypic effects of the absence of a J protein could be rescued by overexpression of only one other J protein, Jjj1, which, like Zuo1, is ribosome-associated. In contrast, the severe growth phenotype caused by the absence of the cytosol's most abundant J protein, Ydj1, was substantially rescued by expression of J domain-containing fragments of many cytosolic J proteins. We conclude that many functions of Hsp70 chaperone machineries only require stimulation of Hsp70's ATPase activity by J protein partners. However, a subset of Hsp70 functions requires specific J protein partners, likely demanding either sublocalization within the compartment or binding to specific client proteins.