Novel regulators of protein quality control and morphogenesis in Schizosaccharomyces pombe

Abstract

The cell wall is a protective barrier and an important morphogenetic element. In most fungi, the main components of the cell wall are chitin, ß-glucans and mannoproteins. Enzymes involved in cell wall construction are membrane- or cell wall- associated proteins that must be delivered to the cell surface in order to exert their function. A great effort has been directed to study the regulation of chitin synthesis, which has led to the characterization of a series of specific regulators. The situation is different in the case of ß-glucans, where practically the only regulators that are under study are the Rho GTPases and their modulators. Our general objective is to study the regulation of the synthesis of ß-glucan by the mechanisms of vesicle transport. A large number of proteins enter the secretory pathway, and about one third of them are unfolded or misfolded when they are synthesized. Cells have quality control mechanisms to detect and correct protein folding defects, and to eliminate proteins that are irreversibly misfolded. The best-known of these mechanisms, UPR and ERAD, act at the endoplasmic reticulum. However, the capacity of these systems is limited, and some non-native proteins can escape from the ER and undergo anterograde transport to the Golgi complex before intracellular disposal. There is growing evidence for post-ER quality control mechanisms in which the escapees either undergo retrograde transport to the ER and are re-targeted to the ERAD or are transported to post-Golgi compartments. In the latter case, the usual final destination for these unfolded proteins is the vacuole, although in some cases they are directed to the cell surface (Arvan, 2002) . In S. pombe, Cfr1p and Csr1p form a complex at the Golgi and are required for glucan synthesis. In the absence of cfr1+, csr1+, or both genes, ¿ and ß glucan synthases are delivered to the cell surface; however, the amount of these proteins is above or below the wild-type level, depending on the position of the HA or GFP tags. These results suggest that minor defects in protein folding have different impact in the cfr1¿ and csr1¿ mutants than in the control strain. This effect on protein stability is dependent on the sensor for unfolded protein stress Ire1p. cfr1¿ and csr1¿ mutants are sensitive to compounds that induce defects in protein folding, and double cfr1¿ ire¿ and cfr1¿ gpt1¿ mutants are more sensitive to these agents than single mutants. Additionally, cfr1¿ and csr1¿ mutants exhibit a dramatic defect in cytokinesis when grown under osmotic stress; this phenotype is absent in cfr1¿ ire1¿ and cfr1¿ spm1/pmk1¿ strains (the latter lacking the kinase for the cell wall integrity pathway). Thus, the Cfr1p/Csr1p complex seems to participate in the response to several stresses. Dni1p and Dni2p are claudin-like proteins required for correct lipid organization at the plasma membrane. dni1+ overexpression produces cell death in a control strain, but not in a ire1¿ strain. In the control strain cell death is accompanied by an abnormal extension of the endoplasmic reticulum. Analysis of the phenotype of dni1+ overexpression will help to understand some aspects of the unfolded protein response.