Characterization of an organelle integrity checkpoint

Cellular 'checkpoints' create a hierarchy between otherwise unrelated cellular events. For example, a delay in DNA replication results in a delay in nuclear division due to the 'DNA replication checkpoints'. They are signal transduction pathways that link these unrelated events, e.g. sensing of a block in DNA replication is transduced by a signalling pathway to inhibit nuclear division. The majority of checkpoints have been identified in the cell cycle. However, additional checkpoints exist, e.g. the morphogenesis checkpoint that responds to perturbations in the cytoskeleton. Based on my previous work I propose that a checkpoint exists that links integrity of an eukaryotic organelle, the endoplasmic reticulum, to differentiation programs that down the line will require an intact endoplasmic reticulum. The purpose of this checkpoint is that only cells that can successfully complete the differentiation process will initiate it. Cells with compromised ER function would die trying to complete the differentiation program. The checkpoint consists of two signalling events, one of which I already characterized in some detail. The second one is less well understand at the moment. The only thing we currently know is that cells lacking upstream elements of the checkpoint are defective in initiating the differentiation program. Further, genes involved in the first signalling activity of this checkpoint are not involved in the second signalling event. From a molecular perspective this gives me the opportunity to uncover a novel signal transduction pathway by studying this second signalling event. I will use a range of genetic experiments in Baker's yeast to identify components of this second signalling pathway of this endoplasmic reticulum integrity checkpoint. Biochemical experiments will then be performed to further understand the role of these genes and proteins in this signalling pathway.

This proposal addresses two new concepts: 1. I hypothesize that organelle integrity checkpoints exist. 2. I hypothesize that a new signal transduction pathway in the eukaryotic unfolded protein response (UPR) exists. This work will advance our understanding of mechanisms of signal integration. The UPR is activated when protein folding in the endoplasmic reticulum (ER) is inhibited. Regulation of all known adaptive responses to ER stress by the UPR requires two genes, IRE1 and HAC1. Ire1 is a bifunctional kinase-endoribonuclease that initiates non-spliceosomal splicing of HAC1 mRNA. I identified two events through which the UPR regulates entry of yeast into meiosis. Meiosis is governed by a transcriptional cascade consisting of IME1, early, middle and late meiotic genes. Ime1p activates transcription of early meiotic genes (EMGs). I showed that Hac1p represses EMG transcription, but had no effect on transcription of IME1. Cells deleted for IRE1 were defective in transcription of IME1. Expression of spliced Hac1p in IRE1 deletion cells had no effect on IME1 mRNA levels, arguing that Ire1p regulates IME1 independent of HAC1. I propose that these two signalling activities of the UPR are an organelle integrity checkpoint. Meiosis requires synthesis of phospholipids and cell wall proteins in the ER. The function of this checkpoint is to delay entry into meiosis when ER function, phospholipid, and cell wall synthesis are perturbed. To gain insight into how Ire1p regulates IME1 and to identify new factors downstream of Ire1p I will use three independent but converging genetic approaches: 1. I will determine if the kinase, RNase, or unknown activity of Ire1p is required for regulation of IME1 using chemical genetics. 2. I will determine the minimal region in the IME1 promoter that is responsive to Ire1p. 3. I will determine if IRE1 signals through a pathway that was already implicated in regulation of IME1, or if the pathway originating at Ire1p is novel.