Transcriptional regulation by inner nuclear membrane proteins

Abstract

All cells sense discrete environmental signals and respond by making appropriate adjustments in patterns of gene expression. To achieve this, signaling pathways translate information received at the cell surface into a transcriptional response. An essential feature of inducible signal transduction pathways is the maintenance of the off state of the gene expression when inducing signals are absent. This thesis addresses the mechanisms that ensure the repressed state of SPS-sensor regulated genes in the yeast Saccharomyces cerevisiae.

Yeast cells use the plasma membrane localized SPS-sensor to sense extracellular amino acids present in their growth environment. In response to amino acids, the SPS-sensor endoproteolytically activates Stp1 and Stp2, two latent cytoplasmic transcription factors with redundant functions. SPS-sensor mediated processing removes cytoplasmic retention motifs within the N-terminal domains of these factors. The shorter activated forms efficiently target to the nucleus, bind promoters of SPS-sensor regulated genes and induce their transcription. Recessive loss-of-function mutations in ASI1, ASI2 and ASI3 (amino acid sensor independent) genes bypass the requirement of a functional SPS-sensor and constitutively induce SPS-sensor regulated gene expression. The recessive nature of the asi mutations suggested that Asi proteins act to negatively regulate SPS-sensor signaling. Interestingly, all three Asi proteins are integral membrane proteins with multiple membrane-spanning segments.

The goal of this thesis has been to elucidate the function of Asi1, Asi2 and Asi3. Genetic and biochemical tests demonstrate that Asi proteins are bona fide constituents of the SPS-sensing pathway. All three Asi proteins localize to the inner nuclear membrane where they function in concert to maintain the latent properties of Stp1 and Stp2 under non-inducing conditions. The data indicate that cytoplasmic retention mechanisms, which prevent nuclear accumulation of latent forms of Stp1 and Stp2, are not completely efficient, and low levels of fulllength forms of Stp1 and Stp2 are able to enter the nucleus. In cells lacking either of the Asi proteins, the unprocessed forms of Stp1 and Stp2 that enter the nucleus are able to bind SPSsensor dependent promoters and inappropriately induce gene expression. Thus, the Asi proteins are required to ensure the repressed state of SPS-sensor signaling in the absence of inducing amino acids by restricting promoter access of full length Stp1 and Stp2. The nuclear factor Dal81 was found to augment the activation potential of Stp1 and Stp2 by enhancing their efficiency to bind SPS-sensor regulated promoters. Thus, Dal81 greatly enhances the sensitivity of the SPS sensing pathway. Strikingly, in cells lacking Dal81, the negative regulatory activity of Asi1, Asi2 and Asi3 is not required to maintain the "off state of SPS-sensor regulated gene expression. In summary, this thesis documents the discovery of a novel role of inner nuclear membrane proteins and illuminate an additional and unanticipated layer of transcriptional control in eukaryotic cells.