Heat shock proteins (HSPs) are expressed by virtually all cells under conditions of high temperature and many other stresses. The alpha- crystallin-related, small (sm) HSPs (15-30 kDa) are ubiquitous among eukaryotes but their structure and function remain poorly understood. Recent in vitro data indicate that the smHSPs may act as a type of molecular chaperone, and in vivo studies suggest they may mediate cytoskeletal rearrangements. A long-term goal of research in my laboratory is to elucidate smHSP structure and function. The proposed research will study smHSPs found in the cytosol of higher plants (PsHSP 18.1 and 17.7) and the homologous smHSP from Saccharomyces cerevisiae (ScHSP26). These and other smHSPs are known to form homo-oligomeric complexes in vivo, but the structure of these complexes is not well-defined. We can routinely isolate mg quantities of highly purified recombinant PsHSPl8.1 and 17.7 in a stable oligomeric form, and have shown that these proteins have chaperone activity in vitro. With the aim of developing a quaternary structure model of the smHSPs, analytical ultracentrifugation, limited proteolysis, and chemical crosslinking will be used to examine purified recombinant Ps HSP 18.1, 17.7, and ScHSP26. We will also continue crystallization trials for X-ray structure determination of PsHSP18.1. Further properties of the smHSPs will be tested by site-directed mutagenesis of proposed structural and functional domains. The assembly and in vitro chaperone activity of the mutant proteins will be tested. In addition, an S. cerevisiae mutant has been isolated which requires elevated ScHSP26 levels for viability. Previously, no function could be ascribed to HSP26 because neither deletion nor over-expression strains of yeast showed alterations in phenotype. Complementation cloning of the mutant gene yielded an already identified, essential gene encoding Uso1p, which is homologous to mammalian p115. Both proteins are involved in intracellular protein transport in the ER-Golgi pathway. This HSP26- dependent mutant will facilitate in vivo functional assays for this class of HSPs, and further studies of the interaction of HSP26 with USO1 are proposed to provide insight into ScHSP26 function.
NIH ExPorter
NIH ExPorter