Academic journal article Genetics

Role of the Unfolded Protein Response Pathway in Secretory Stress and Regulation of INO1 Expression in Saccharomyces Cerevisiae

Academic journal article Genetics

Role of the Unfolded Protein Response Pathway in Secretory Stress and Regulation of INO1 Expression in Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

The unfolded protein response pathway (UPR) enables the cell to cope with the buildup of unfolded proteins in the endoplasmic reticulum (ER). UPR loss-of-function mutants, hac1Δ and ire1Δ, are also inositol auxotrophs, a phenotype associated with defects in expression of INO1, the most highly regulated of a set of genes encoding enzymes of phospholipid metabolism. We now demonstrate that the UPR plays a functional role in membrane trafficking under conditions of secretory stress in yeast. Mutations conferring a wide range of membrane trafficking defects exhibited negative genetic interaction when combined with ire1Δ and hac1Δ. At semipermissive temperatures, carboxypeptidase Y transit time to the vacuole was slower in Sec^sup -^ cells containing an ire1Δ or hac1Δ mutation than in Sec^sup -^ cells with an intact UPR. The UPR was induced in Sec^sup -^ cells defective in subcellular membrane trafficking events ranging from ER vesicle trafficking to distal secretion and in erg6Δ cells challenged with brefeldin A. However, the high levels of UPR induction observed under these conditions were not correlated with elevated INO1 expression. Indeed, many of the Sec^sup -^ mutants that had elevated UPR expression at semipermissive growth temperatures failed to achieve wild-type levels of INO1 expression under these same conditions.

THE unfolded protein response pathway (UPR) is a stress response pathway that is activated when unfolded proteins accumulate in the endoplasmic reticulum (ER; COX et al. 1993; COX and WALTER 1996; MORI et al. 1992, 1993). In yeast, the UPR consists of three components: Ire1p, Hac1p, and Rlg1p. Ire1p is a unique ER transmembrane spanning protein kinase/endoribo-nuclease. Hac1p is a transcription factor that is required for expression of UPR-responsive genes, including protein-folding chaperones, such as Kar2p (BiP; KOHNO et al. 1993; MORI et al. 1992, 1993; NIKAWA and YAMASHITA 1992). When improperly folded proteins accumulate in the ER, Ire1p autophosphorylates, thereby activating the Ire1p endoribonuclease activity, which catalyzes the splicing of the HAC1 mRNA (COX and WALTER 1996; MORI et al. 2000), followed by ligation by Rlg1p, a tRNA ligase (SIDRAUSKI et al. 1996). Since only the spliced form of HAC1 mRNA is effectively translated (CHAPMAN and WALTER 1997; KAWAHARA et al. 1997), this regulated splicing leads to expression of Hac1p and subsequent activation of transcription of genes such as KAR2, containing the unfolded protein-responsive element (UPRE) in their promoters.

Cells carrying ire1Δ, hac1Δ, or rlg1-100 mutations are sensitive to drugs such as tunicamycin, which causes accumulation of misfolded proteins in the ER (NIKAWA et al. 1996; SHAMU and WALTER 1996; SIDRAUSKI et al. 1996; COX et al. 1997). In addition, ire1Δ, hac1Δ, or rlg1-100 mutants are inositol auxotrophs (NIKAWA and YAMASHITA 1992; COX et al. 1993; NIKAWA et al. 1996; SIDRAUSKI et al. 1996), a phenotype associated with defects in expression of genes related to phospholipid metabolism, especially INO1, the structural gene encoding myo-inositol 3-phosphate synthase (for review see HENRY and PATTON-VOGT 1998). INO1 and coregulated genes of phospholipid metabolism contain the inositol-sensitive upstream activating sequence (UAS^sub INO^) repeated element in their promoters and exhibit complex transcriptional regulation in response to a variety of environmental factors including the availability of soluble precursors of phospholipid metabolism such as inositol (CARMAN and HENRY 1999). Wild-type yeast cells express INO1 and other UAS^sub INO^-containing genes at a high level when inositol is limiting in the growth medium and repress these same genes when inositol is plentiful (HIRSCH and HENRY 1986; GREENBERG and LOPES 1996; CARMAN and HENRY 1999; LOEWEN et al. 2004). COX et al. (1997) reported that the UPR is activated in the absence of inositol and suggested that the activation of the UPR might be directly involved in the mechanism by which INO1 transcription is activated when inositol is limiting. …

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