A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.

SUMMARY The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast.

Abstract. In Saccharomyces cerevisiae the vacuolar protein aminopeptidase I (API) is localized to the vacuole independent of the secretory pathway. The alternate targeting mechanism used by this protein has not been characterized. API is synthesized as a 61-kD soluble cytosolic precursor. Upon delivery to the vacuole, the amino-terminal propeptide is removed by proteinase B (PrB) to yield the mature 50-kD hydrolase. We exploited this delivery-dependent maturation event in a mutant screen to identify genes whose products are involved in API targeting. Using antiserum to the API propeptide, we isolated mutants that accumulate precursor API. These mutants, designated cvt, fall into eight complementation groups, five of which define novel genes. These five complementation groups exhibit a specific defect in maturation of API, but do not have a significant effect on vacuolar protein targeting through the secretory pathway. Localization studies show that precursor API accumulates outside of the vacuole in all five groups, indicating that they are blocked in API targeting and/or translocation. Future analysis of these gene products will provide information about the subcellular components involved in this alternate mechanism of vacuolar protein localization.

The vacuolar protein aminopeptidase I (API) uses a novel cytoplasm-to-vacuole targeting (Cvt) pathway. Complementation analysis of yeast mutants defective for cytoplasm-to-vacuole protein targeting (cvt) and autophagy (apg) revealed seven overlapping complementation groups between these two sets of mutants. In addition, all 14 apg complementation groups are defective in the delivery of API to the vacuole. Similarly, the majority of nonoverlapping cvt complementation groups appear to be at least partially defective in autophagy. Kinetic analyses of protein delivery rates indicate that autophagic protein uptake is induced by nitrogen starvation, whereas Cvt is a constitutive biosynthetic pathway. However, the machinery governing Cvt is affected by nitrogen starvation as targeting defects resulting from API overexpression can be rescued by induction of autophagy.The vacuole/lysosome is the major hydrolytic compartment of the cell and as such is central to survival during nitrogen starvation (1). When cells sense nitrogen starvation, cytoplasmic proteins and organelles are packaged nonselectively into autophagosomes, which are then targeted to the vacuole for degradation and turnover of their constituent components by resident hydrolases (2, 3). In this manner, amino acids as well as nucleic acids can be recycled to enable stress survival. In yeast, this autophagic process has been studied primarily through microscopic analysis. During autophagy, large doublemembraned vesicles (500 nm) nonselectively surround cytoplasmic proteins and organelles (2, 4). On delivery to the vacuole, the outermost membrane is proposed to fuse to the vacuolar membrane, resulting in the delivery of a still intact vesicle (autophagic body) into the vacuolar lumen (2, 5). These autophagic bodies are then degraded in a subsequent step that depends on proteinase B (4). Although two collections of autophagy mutants have been identified (6, 7), the molecular details of this process have not yet been elucidated.The resident hydrolases that mediate the digestive capacity of the vacuole are delivered to this organelle by two distinctly different protein transport pathways. The majority of vacuolar proteins are targeted through the secretory pathway (reviewed in refs. 8 and 9), whereas at least one vacuolar protein, aminopeptidase I (API) (10), uses the cytoplasm-to-vacuole targeting (Cvt) pathway. In mammalian cells, decreasing lumenal pH through the endocytic compartments is proposed to mediate the receptor/ligand dissociation reactions required for correct sorting through these compartments (11). Early evidence suggests that the same mechanism may mediate sorting in yeast as well; one important feature of the vacuolar membrane is the presence of a vacuolar ATPase (V-ATPase), which uses ATP to transport protons into the lumen. Maintenance of the resulting ApH is essential not only for optimal activity of many vacuolar proteases but also for efficient delivery of soluble vacuolar proteins by the secretory (12-14) and Cvt (15) pathways.The ...

Hsp70s mediate protein folding, translocation, and macromolecular complex remodeling reactions. Their activities are regulated by proteins that exchange ADP for ATP from the nucleotide-binding domain (NBD) of the Hsp70. These nucleotide exchange factors (NEFs) include the Hsp110s, which are themselves members of the Hsp70 family. We report the structure of an Hsp110:Hsc70 nucleotide exchange complex. The complex is characterized by extensive protein:protein interactions and symmetric bridging interactions between the nucleotides bound in each partner protein's NBD. An electropositive pore allows nucleotides to enter and exit the complex. The role of nucleotides in complex formation and dissociation, and the effects of the protein:protein interactions on nucleotide exchange, can be understood in terms of the coupled effects of the nucleotides and protein:protein interactions on the open-closed isomerization of the NBDs. The symmetrical interactions in the complex may model other Hsp70 family heterodimers in which two Hsp70s reciprocally act as NEFs.