Network of general and specialty J protein chaperones of the yeast cytosol

Abstract

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 domaincontaining 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.Hsp40 ͉ Hsp70 ͉ molecular chaperone ͉ multigene family T hrough their action in protein folding, degradation, translocation across membranes, and disassembly of protein complexes, molecular chaperones are important participants in many crucial cellular processes (1, 2). Hsp70s and their J protein partners (at times referred to as Hsp40s) constitute an important component of the cellular ''chaperone'' in virtually all living systems (3). In most cellular compartments, an Hsp70 has multiple J protein partners. The cytosol, a hub of activity for a variety of crucial cellular processes, is no exception. With a goal of better understanding the degree of functional overlap among J proteins, as well as their individual specificities, we choose the cytosol of Saccharomyces cerevisiae as the focus of a comprehensive analysis of J protein function.Although J proteins are obligate partners of Hsp70s, it is the Hsp70 that has long been considered the core of the Hsp70:J protein chaperone machine by virtue of their client protein interaction, which is modulated by nucleotide binding (4). ATP binding fosters rapid binding to client proteins, whereas nucleotide hydrolysis stabilizes the interaction. The cytosol of S. cerevisiae contains two predominant classes of Hsp70s, Ssa (SSA1-4) and Ssb (SSB1-2) (5). Yeast cells expressing only one representative of a class grow very similarly to wild-type cells under a variety of conditions. But the classes are functionally distinct, because a member of the Ssa class cannot substitute for an Ssb Hsp70 and vice versa (6).A role of all J proteins is stimulation of the ATPase activity of their partner Hsp70s. Such stimulation...

The Mammalian Hsp40 ERdj3 Requires Its Hsp70 Interaction and Substrate-binding Properties to Complement Various Yeast Hsp40-dependent Functions

“…To better resolve this apparent discrepancy, we have compared the abilities of two type I JDPs, a mammalian, ER-lumenal protein (ERdj3), and a yeast cytoplasmic protein (Ydj1) to functionally substitute for each other. Our data, combined with those of a previous study (13), suggest that both the binding of JDPs to substrates and their association with Hsp70s are essential to support cell viability and chaperone-dependent function.…”

“…YDJ1 deletion results in slow growth at 30°C and inviability at elevated temperatures (44). However, the slow growth of the ydj1⌬ strain can be rescued by the overexpression of at least five other cytosolic JDPs (13). Interestingly, expression of the J domain alone of these five JDPs is sufficient to substitute for Ydj1 at 30°C, suggesting that the J domain-mediated activation of Hsp70 ATPase activity is critical to support the growth of ydj1⌬ yeast (13).…”

“…Notably, the number of JDPs exceeds the number of Hsp70s and nucleotide exchange factors in most organisms/organelles. In fact, a single Hsp70 can interact with different JDPs to form unique Hsp70-JDP pairs that participate in specific cellular functions (12,13). Despite the high conservation of the J domain, JDPs are not necessarily interchangeable between organelles or organisms (11,14), further suggesting that there is specificity in the interaction between Hsp70s and JDPs.…”

“…To begin to address this hypothesis, a recent study tested the ability of 13 different JDPs to interact with resident Hsp70s in the yeast cytoplasm (13). In many cases, the ability of a JDP to stimulate the ATPase activity of a particular Hsp70 was sufficient to constitute a functional pair.…”

Identification of potential HLA class I and class II epitope precursors associated with heat shock protein 70 (HSPA)

“…Although most of the peptides showed affinity to HSPA on the peptide array, a number of peptides were defined as non-binding. This potentially was caused by the lack of HSPA cofactors, such as DNAJ proteins, which are present in the cell and dictate the binding characteristic of HSPA (Cheetham and Caplan 1998;Johnson and Craig 2001;Sahi and Craig 2007). On the other hand, low-affinity binding peptides could be considered as contaminants; however, peptides C1, C2, C3, C5 and C6, which showed a low level of binding, contain common sequences with peptides which have been previously reported as co-purified with mouse HSPA (Grossmann et al 2004; Table 1).…”