Import into and Degradation of Cytosolic Proteins by Isolated Yeast Vacuoles

In eukaryotic cells, both lysosomal and nonlysosomal pathways are involved in degradation of cytosolic proteins. The physiological condition of the cell often determines the degradation pathway of a specific protein. In this article, we show that cytosolic proteins can be taken up and degraded by isolated Saccharomyces cerevisiae vacuoles. After starvation of the cells, protein uptake increases. Uptake and degradation are temperature dependent and show biphasic kinetics. Vacuolar protein import is dependent on cytosolic heat shock proteins of the hsp70 family and on protease-sensitive component(s) on the outer surface of vacuoles. Degradation of the imported cytosolic proteins depends on a functional vacuolar ATPase. We show that the cytosolic isoform of yeast glyceraldehyde-3-phosphate dehydrogenase is degraded via this pathway. This import and degradation pathway is reminiscent of the protein transport pathway from the cytosol to lysosomes of mammalian cells.

Pharmacologic shifting of a balance between protein refolding and degradation mediated by Hsp90

The role of the abundant stress protein Hsp90 in protecting cells against stress-induced damage is not well understood. The recent discovery that a class of ansamycin antibiotics bind specifically to Hsp90 allowed us to address this problem from a new angle. We find that mammalian Hsp90, in cooperation with Hsp70, p60, and other factors, mediates the ATP-dependent refolding of heat-denatured proteins, such as firefly luciferase. Failure to refold results in proteolysis. The ansamycins inhibit refolding, both in vivo and in a cell extract, by preventing normal dissociation of Hsp90 from luciferase, causing its enhanced degradation. This mechanism also explains the ansamycin-induced proteolysis of several protooncogenic protein kinases, such as Raf-1, which interact with Hsp90. We propose that Hsp90 is part of a quality control system that facilitates protein refolding or degradation during recovery from stress. This function is used by a limited set of signal transduction molecules for their folding and regulation under nonstress conditions. The ansamycins shift the mode of Hsp90 from refolding to degradation, and this effect is probably amplified for specific Hsp90 substrates.

The Mos/mitogen-activated protein kinase (MAPK) pathway regulates the size and degradation of the first polar body in maturing mouse oocytes.

Mos is an upstream activator of mitogen-activated protein kinase (MAPK) and, in mouse oocytes, is responsible for metaphase II arrest. This activity has been likened to its function in Xenopus oocytes as a component of cytostatic factor. Thus, Mos-deficient female mice (MOS-/-) are less fertile and oocytes derived from these animals fail to arrest at metaphase II and undergo parthenogenetic activation [Colledge, W. H., Carlton, M. B. L., Udy, C. B. & Evans, M. J. (1994) Nature (London) 370, 65-68 and Hashimoto, N., Watanabe, N., Furuta. Y., Tamemoto, B., Sagata, N., Yokoyama, M., Okazaki, K., Nagayoshi, M., Takeda, N., Ikawa, Y. & Aizawa, S. (1994) Nature (London) 370, 68-71]. Here we show that maturing MOS-/- oocytes fail to activate MAPK throughout meiosis, while p34cdc2 kinase activity is normal until late in metaphase II when it decreases prematurely. Phenotypically, the first meiotic division of MOS-/- oocytes frequently resembles mitotic cleavage or produces an abnormally large polar body. In these oocytes, the spindle shape is altered and the spindle fails to translocate to the cortex, leading to the establishment of an altered cleavage plane. Moreover, the first polar body persists instead of degrading and sometimes undergoes an additional cleavage, thereby providing conditions for parthenogenesis. These studies identify meiotic spindle formation and programmed degradation of the first polar body as new and important roles for the Mos/MAPK pathway.