An atlas of chaperone–protein interactions in Saccharomyces cerevisiae : implications to protein folding pathways in the cell

Abstract

Molecular chaperones are known to be involved in many cellular functions, however, a detailed and comprehensive overview of the interactions between chaperones and their cofactors and substrates is still absent. Systematic analysis of physical TAP‐tag based protein–protein interactions of all known 63 chaperones in Saccharomyces cerevisiae has been carried out. These chaperones include seven small heat‐shock proteins, three members of the AAA+ family, eight members of the CCT/TRiC complex, six members of the prefoldin/GimC complex, 22 Hsp40s, 1 Hsp60, 14 Hsp70s, and 2 Hsp90s. Our analysis provides a clear distinction between chaperones that are functionally promiscuous and chaperones that are functionally specific. We found that a given protein can interact with up to 25 different chaperones during its lifetime in the cell. The number of interacting chaperones was found to increase with the average number of hydrophobic stretches of length between one and five in a given protein. Importantly, cellular hot spots of chaperone interactions are elucidated. Our data suggest the presence of endogenous multicomponent chaperone modules in the cell. ### Synopsis Molecular chaperones are defined as a group of highly interactive proteins that modulate the folding and unfolding of other proteins, or the assembly and disassembly of protein–protein, protein–DNA, and protein–RNA complexes ([Hartl and Hayer‐Hartl, 2002][1]; [Deuerling and Bukau, 2004][2]; [Saibil, 2008][3]). In addition, chaperones are known to be involved in many cellular processes and pathways such as protein translocation across membranes, ribosomal RNA processing, and endoplasmic reticulum associated protein degradation (ERAD). A well‐studied and well‐defined model organism like Saccharomyces cerevisiae (budding yeast) has a total of 63 chaperones: seven small heat‐shock proteins, three chaperones of the AAA+ family, eight of the CCT/TRiC complex, six of the prefoldin/GimC complex, 22 Hsp40s, one Hsp60, 14 Hsp70s, and two Hsp90s ([Sghaier et al , 2004][4]). These represent 50 chaperones/chaperone complexes. We experimentally obtained and analyzed the physical interaction network of all 50 chaperones/chaperone complexes in yeast based on TAP‐tag interactions. The presence of molecular chaperones in complexes obtained from the tandem affinity purification of 4562 different endogenously TAP‐tagged proteins in yeast cells was determined by mass spectrometry. A total of 21 687 unique pairs of interactions were identified as high confidence. These interactions are between 63 chaperones and a total of 4340 other proteins; in addition, there are 259 chaperone–chaperone interactions ([Figure 1][5]). A correlation analysis with the number of interacting chaperones showed several protein properties that influence the propensity of proteins to interact with chaperones: (1) proteins that have a larger number of hydrophobic stretches of length between 1 and 5 interact with more chaperones; (2) however, more hydrophobic proteins interact with fewer chaperones whereas more hydrophilic proteins interact with more chaperones; (3) larger proteins and multi‐domain proteins interact with more chaperones than smaller and simpler proteins; (4) proteins enriched in the charged residues Asp, Glu, and Lys also interact with more chaperones. Analysis of the half‐lives of chaperone interactors yielded an important observation. Our data indicate that the number of chaperone interactors does not correlate with in vivo protein turnover. Hence, although chaperones might work to stabilize proteins, they do not directly affect their half‐lives. Hence, the major determinants of protein half‐life in the cell might reside with the degradation machinery. Molecular chaperones seem to be especially important for the maintenance of protein complexes and pathways that are closely associated with nuclear activities. This strongly indicates an important role for the chaperone systems in maintaining genomic stability and gene expression. In addition to interactions of chaperones with other proteins, we also detected extensive TAP‐tagged protein–protein interactions between different chaperones, suggesting the prevalence of functional cooperation, physical association, or functional redundancy between individual chaperones. There are 195 such chaperone–chaperone interactions between the 50 chaperones/chaperone complexes. To elucidate significantly important two‐chaperone relationships based on interactor overlap, the numbers of shared interactors for each pair of chaperones were counted and a Z ‐score was calculated to determine enrichment for common interactors. Chaperone pairs with a Z ‐score greater than or equal to two were considered to form a statistically significant functional module. Among the 50 chaperones/chaperone complexes, 41 out of a total of 1079 chaperone pairs with overlapping interactors were found to be significant ([Figure 6A and B][6]). Most of the modules were found within the cytoplasm/nucleus, but four modules were found between Ssa1, Ssa2, Ssb1 and Sse1, which are located in the cytoplasm/nucleus, and the mitochondrial chaperone Hsp78. It is interesting to note that 38 out of the 41 chaperone modules are also found in the 195 chaperone–chaperone TAP‐based interactions. As it is known that more than two chaperones might be involved in mediating the correct folding or stability of substrate proteins, significant modules composed of 3–5 individual chaperones were also derived using a similar procedure as for the two‐component modules. The most frequently observed larger modules contain the chaperones of the Hsp40 and Hsp70 family members or of the Hsp90 and Hsp70 members ([Figure 6B][6]). This is consistent with our current detailed understanding of the biochemistry of chaperone function:...