To maintain the integrity and identity of membrane-bound compartments, membrane fusion is promoted by fusases in a non-leaky manner. During yeast mating, two partners of a mating pair must fuse their cell membranes. The multipass membrane protein Prm1p promotes this extracytoplasmic fusion event. Less than half of prm1 x prm1 mating pairs successfully fuse, instead arresting at the step of membrane fusion or undergoing simultaneous cell lysis. prm1 x prm1 mating pair lysis is enhanced when extracellular Ca2+ is removed. As revealed by time-lapse microscopy, mating pair fusion and lysis events initiate with identical kinetics. Furthermore, cytoplasmic continuity is generated concurrent with cell lysis. These results suggest that cell lysis is linked to the mechanism of cell fusion. Prm1p promotes cell fusion and prevents cell lysis as a covalently linked homodimer. Substitution of cys-120 and cys-545 prevents covalent dimerization but does not decrease association of Prm1p monomers. Mutants unable to form the covalent link between dimers are severely compromised for Prm1p activity. PRM1 mutants show a unique genetic interaction with KEX2, a gene encoding a late-Golgi protease. Though prm1 x wt and wt x kex2 (MATa x MATα) mating pairs do not have strong mating defects, prm1 x kex2 mating pairs fuse with less than 50% efficiency. Unfused wt x kex2 mating pairs exhibit membrane enclosed structures embedded in the cell wall separating mating partners ("blebs"). When PRM1 is deleted, some prm1 x prm1kex2 mating pairs extend protrusions into the space of the partner cell that are apparently devoid of cytoplasmic contents ("enormous barren bubbles"). Another mating-induced membrane protein, Fig1p, is required for efficient cell membrane fusion during yeast mating. fig1 x fig1 mating pairs have a mild bilateral fusion defect, and 10% of unfused mating pairs arrest at the step of membrane fusion. PRM1, FIG1, and KEX2 all act during late steps of yeast mating; however, mutants of both KEX2 and FIG1 are pleiotropic and are likely to influence membrane fusion indirectly. In contrast, Prm1p is intimately involved in controlling the fidelity of membrane fusion and may constitute a part of the yeast cell fusion machinery.

Abstract

In eukaryotic cells, the untoward accumulation of misfolded proteins inside the endoplasmic reticulum (ER) triggers a transcriptional program that restores cellular homeostasis. This program, called the unfolded protein response (UPR), is activated through a series of enzymatic steps that culminate in the production of a transcription factor that upregulates a set of target genes required for cell survival during ER stress. Activation occurs through an ER-resident sensor, Ire1, which is stimulated by misfolded proteins in the ER lumen. Activated Ire1 transmits a signal to its cytosolic portion, containing a kinase and an RNase, which trans-autophosphorylates and activates the RNase to cleave an mRNA encoding the Hac1 transcription factor. The cleavage products are ligated by tRNA ligase, Trl1, to produce a spliced form of HAC1 mRNA that is translated into the transcription factor. Together, the coordinated activation of Ire1 and ligation by Trl1 to produce spliced HAC1 mRNA comprise the central mechanism of UPR activation. This thesis describes my work toward understanding two key regulatory steps in UPR activation.

Several lines of evidence suggest that Ire1 kinase plays a role in the UPR that is autonomous from its role in RNase activation. First, RNase L, a close homolog of Ire1, is an RNase with a pseudokinase domain that has lost activity through evolution. Conservation of Ire1's kinase suggests that activity is important for UPR fitness. Moreover, in vitro studies have shown that kinase-inactive Ire1 retains RNase activity and, in vivo, kinase activity can be bypassed by ATP-competitive drugs. In an endeavor to understand the importance of Ire1 kinase we discovered that kinase activity contributes to the attenuation of Ire1 signaling and is imperative for homeostatic adaptation during ER stress.

The final chapters of this thesis describe my work to decipher how tRNA ligase achieves pathway specificity for the UPR. Trl1 splits its time between in HAC1 mRNA splicing and in tRNA splicing. Our efforts to discern how Trl1 acts in HAC1 mRNA splicing were unfruitful, however, this work enlightened our knowledge of Trl1 biology and set the stage for future work to elucidate Trl1's role in the UPR.