Hsp70 and J-protein Multigene Families: Generalists and Specialists

Complementary to our studies of molecular chaperones in particular cellular processes we aim to understand the basis of functional differences amongst molecular chaperones. Unlike many molecular chaperones, Hsp70s-based chaperone machines are encoded by sizeable multigene families. We focus on the J-protein component of Hsp70-based machinery, as J-protein partners orchestrates the ability of Hsp70 to participate in a wide array of complex and diverse biological processes.

J-proteins are obligate co-chaperones of Hsp70s, stimulating their ATPase activity, and thus allowing them to function in multiple important cellular processes. In most cellular compartments an Hsp70 works with multiple, structurally divergent J-proteins, which may have only the J-domain, the domain responsible for ATPase stimulation in common. Our goal is to understand the functional diversity and the mechanistic basis of such specificity. For example, to better understand the functional specificity of J-proteins and the complexity of the Hsp70:J-protein network, we undertook a comprehensive analysis of the 13 J-proteins present in the yeast cytosol. The results of this analysis led us to classify J-proteins into “specialist” and “generalist” categories.

Specialists: Phenotypes caused by the absence of four, Sis1, Jjj1, Jjj3 and Cwc23, could not be rescued by over-expression of any other cytosolic J-protein, demonstrating the distinctive qualities of these J-proteins. In one case, that of ribosome-associated Zuo1, the phenotypic effects of the absence of the J-protein could be rescued by only one other J-protein, Jjj1, which is also associated with the 60S ribosomal subunit.

Generalists: In contrast to this obvious specialization, 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 other cytosolic J-proteins. We conclude that many cellular functions of Hsp70 chaperone machineries only require stimulation of the ATPase activity by J-protein partners.

Questions we are addressing concerning the specialization of J-proteins include:

• Specialized role of Jjj1 in ribosome biogenesis. The J-protein Jjj1 is associated with 60S ribosome particles and plays an important role in a late step in biogenesis of the subunit in the cytosol. Cells lacking Jjj1 have phenotypes very similar to those of cells lacking Rei1, a ribosome biogenesis factor associated with pre-60S ribosomal particles in the cytosol. In cells lacking Rei1 or Jjj1 two other biogenesis factors, among the last to be removed prior to generation of mature subunits, remain bound to 60S subunits. How does Jjj1 and its Hsp70 partner Ssa mechanistically act to promote dissociation of these factors?

Jjj1 is normally approximately 40 fold less abundant than Zuo1, a ribosome-associated J-protein important in chaperoning newly synthesized polypeptides as they exit the ribosome. Overexpression of Jjj1 partially rescues the phenotypes of cells lacking Zuo1. Does Jjj1 normally play an additional role as a chaperone for nascent chains?

• Specialized role of Sis1 in prion propagation. The function of the J-protein Sis1 and the Hsp70 Ssa is required for the yeast prion [RNQ+] to be propagated. The closely related J-protein Ydj1 will not suffice. These facts raise two fundamental questions: Why is a chaperone required for prion maintenance? What special features does the Sis1 J-protein possess that other J proteins do not? Remarkably, the human ortholog of Sis1 is capable of propagation of [RNQ+], but the ortholog of Ydj1 is not. We have mapped the functional difference between Sis1 and Ydj1 to the glycine-rich region adjacent to the J domain. What is the function of the G/F region? Is it important for targeting Rnq1 to Hsp70s or for modulating Hsp70’s reaction cycle?

Selected Craig lab publications:

Higurashi, T., Hines, J.K., Sahi, C., Aron, R., and Craig E.A. (2008) Specificity of the J-Protein SIS1 in the Propagation of Three Yeast Prions. Proc Natl Acad Sci USA. 105:16596-16601. [PDF]

Sahi, C. and Craig, E.A. (2007) Network of general and specialty J protein chaperones of the yeast cytosol. Proc Natl Acad Sci USA. 104(17):7163-7168. [PDF]

Meyer, A.E., Hung, N.J., Yang, P., Johnson, A.W., Craig, E.A. (2007) The specialized cytosolic J-protein, Jjj1, functions in 60S ribosomal subunit biogenesis. Proc Natl Acad Sci USA. 104:1557-1563. [PDF]

Lopez, N, Aron, R and Craig, EA (2003) Specificity of the Class II Hsp40 Sis1 in maintenance of the yeast prion [RNQ+] Mol. Biol. Cell 14:1172-81. [PDF]