We have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro. Although the recovery of transferrin binding activity in exosomes is at best approximately 25-30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I-labelled transferrin receptor is measured using an that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75-80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H-nitrobenzylthioinosine binding to measure the nucleoside transporter. These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling. We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.

Mammalian cells coexpress a family of heat shock factors (HSFs) whose activities are regulated by diverse stress conditions to coordinate the inducible expression of heat shock genes. Distinct from HSF1, which is expressed ubiquitously and activated by heat shock and other stresses that result in the appearance of nonnative proteins, the stress signal for HSF2 has not been identified. HSF2 activity has been associated with development and differentiation, and the activation properties of HSF2 have been characterized in hemintreated human K562 erythroleukemia cells. Here, we demonstrate that a stress signal for HSF2 activation occurs when the ubiquitin-proteasome pathway is inhibited. HSF2 DNA-binding activity is induced upon exposure of mammalian cells to the proteasome inhibitors hemin, MG132, and lactacystin, and in the mouse ts85 cell line, which carries a temperature sensitivity mutation in the ubiquitin-activating enzyme (E1) upon shift to the nonpermissive temperature. HSF2 is labile, and its activation requires both continued protein synthesis and reduced degradation. The downstream effect of HSF2 activation by proteasome inhibitors is the induction of the same set of heat shock genes that are induced during heat shock by HSF1, thus revealing that HSF2 affords the cell with a novel heat shock gene-regulatory mechanism to respond to changes in the protein-degradative machinery.The cellular response to stresses such as heat shock is tightly controlled at the level of transcription, and in larger eukaryotes it is mediated by a family of heat shock transcription factors (HSFs) corresponding to HSF1 through HSF4 (37, 38, 65), which recognize and bind to heat shock elements (HSEs) present in the promoter regions of heat shock genes (11). The expression of multiple HSF family members in larger eukaryotes endows the cell with a mechanism to sense and respond to diverse forms of stress. HSF1 and HSF3 are activated following exposure to traditional forms of environmental and physiological stress such as heat shock and chemical stress (37,38,41,65). In avian cells expressing HSF1 but in which the HSF3 gene is deleted, the heat shock response is strongly diminished, which reveals a new level of regulatory interaction among members of the HSF family (57). The suggestion that HSFs may exhibit complex interactions with other transcription factors is further demonstrated by the observation that HSF3 expressed in avian cells can be activated in the absence of stress by direct protein-protein interaction with the DNA binding domain of the c-Myb proto-oncogene (27).Another member of the HSF family, HSF2, is 40% related in sequence to HSF1 and HSF3, with the regions of highest sequence conservation corresponding to the DNA-binding and heptad repeat regions. However, unlike HSF1 and HSF3, HSF2 is not activated in response to heat shock and most other forms of cellular stress (37, 38, 65). HSF2 has been described as having properties of a development-and differentiationassociated transcription factor, in part due to observatio...

Changes in protein levels and the folded states of proteins are sensed by molecular chaperones and the degradative machinery that function to maintain cellular protein homeostasis. The importance of these systems is highlighted by their high degree of functional and structural conservation across evolution, from prokaryotes to eukaryotes, and their essential roles in a variety of cellular functions.1-3 The chaperone network, proteases, and components of the degradative machinery are also linked by common genetic regulatory pathways. For example, many of the proteins involved in protein folding and degradation are also heat-shock proteins whose expression is induced when cells are stressed and accumulate denatured or malfolded proteins.1,3-6 The family of heat-shock proteins (HSPs) encompasses a number of functionally related proteins that are expressed constitutively and/or at elevated levels upon exposure of cells to a variety of stress conditions including elevated temperature, arsenite, heavy metals, amino acid analogues, and oxidants. 1,4,6 The chaperones and proteolytic systems often function together to determine the ultimate fates of proteins and are often coordinately regulated, as outlined in FIGURE 1. The heat-shock response regulates proteolysis by enhancing the expression of proteolytic factors and proteases, as well as of chaperones which may function as cofactors for proteolysis.2,3,5 The stress response in both prokaryotes and eukaryotes is, in turn, regulated by the proteolytic machinery 7-10 (also A. Mathew, S. K. Mathur, and R. I. Morimoto, unpublished observations). This review will address the chaperone requirements in eukaryotic proteolysis and the regulation of eukaryotic chaperone expression by the ubiquitin-proteasome proteolytic system. A number of recent reviews have also addressed chaperone activities associated with proteolysis in prokaryotes and eukaryotes. 3,11,12 BIOCHEMICAL PROPERTIES OF MOLECULAR CHAPERONESChaperones are involved at various stages in protein biogenesis, regulating their structure and function under normal physiological conditions, as well as during and following stress which results in protein unfolding and misfolding. The family of heat-shock proteins and molecular chaperones includes Hsp100, Hsp90, Hsp70 (dnaK), Hsp60 (GroEL), Hsp40 (dnaJ), and the small HSPs. These chaperones are abundant and ubiquitous and function constitutively in protein synthesis and folding, protein translocation into membrane compartments, and the assembly and disassembly of oligomers. 2,13 Chaperones often function in concert with other 99 a

Vertebrate cells express a family of heat shock transcription factors (HSF1 to HSF4) that coordinate the inducible regulation of heat shock genes in response to diverse signals. HSF1 is potent and activated rapidly though transiently by heat shock, whereas HSF2 is a less active transcriptional regulator but can retain its DNA binding properties for extended periods. Consequently, the differential activation of HSF1 and HSF2 by various stresses may be critical for cells to survive repeated and diverse stress challenges and to provide a mechanism for more precise regulation of heat shock gene expression. Here we show, using a novel DNA binding and detection assay, that HSF1 and HSF2 are coactivated to different levels in response to a range of conditions that cause cell stress. Above a low basal activity of both HSFs, heat shock preferentially activates HSF1, whereas the amino acid analogue azetidine or the proteasome inhibitor MG132 coactivates both HSFs to different levels and hemin preferentially induces HSF2. Unexpectedly, we also found that heat shock has dramatic adverse effects on HSF2 that lead to its reversible inactivation coincident with relocalization from the nucleus. The reversible inactivation of HSF2 is specific to heat shock and does not occur with other stressors or in cells expressing high levels of heat shock proteins. These results reveal that HSF2 activity is negatively regulated by heat and suggest a role for heat shock proteins in the positive regulation of HSF2.The heat shock response is a cellular defense against the deleterious effects of physiological and environmental stress that is mediated by heat shock transcription factors (HSFs). In vertebrates, four members of the HSF family have been identified (27,28,38,40,46), and of these, HSF1 and HSF2 are ubiquitously expressed and conserved (27,40). HSF1 and HSF2 are expressed as inert monomers and dimers, respectively, in unstressed cells. Upon activation, these HSFs trimerize and function as transcriptional activators that bind with similar, but not identical, specificities to the heat shock element (HSE), thus regulating heat shock gene transcription and hence the expression of diverse heat shock proteins and molecular chaperones (2,40,49,62).HSF1 and HSF2 differ in their pathways of activation and the nature of their transcriptional responses. Whereas HSF1 is activated upon exposure to a multitude of physiological and environmental stresses (41, 62), HSF2 activity appears to be more selective, being induced upon down-regulation of the ubiquitin-proteasome pathway (22), during differentiation (36,37,42,49), and in early development (9, 23, 37). Comparison of the HSF1 and HSF2 transactivation domains as chimeric proteins with a minimal heterologous DNA binding domain reveals that HSF1 is a more potent transcriptional activator than HSF2 (47, 59, 60). These observations are also supported by studies comparing hsp70 and hsp90 gene transcription rates in K562 cells in which endogenous HSF1 or HSF2 was activated (49). In those studies, HSF1 ...