Asi1 is an inner nuclear membrane protein that restricts promoter access of two latent transcription factors

Stp1 and Stp2 are homologous transcription factors in yeast that are synthesized as latent cytoplasmic precursors with NH2-terminal regulatory domains. In response to extracellular amino acids, the plasma membrane–localized Ssy1–Ptr3–Ssy5 (SPS) sensor endoproteolytically processes Stp1 and Stp2, an event that releases the regulatory domains. The processed forms of Stp1 and Stp2 efficiently target to the nucleus and bind promoters of amino acid permease genes. In this study, we report that Asi1 is an integral component of the inner nuclear membrane that maintains the latent characteristics of unprocessed Stp1 and Stp2. In cells lacking Asi1, full-length forms of Stp1 and Stp2 constitutively induce SPS sensor–regulated genes. The regulatory domains of Stp1 and Stp2 contain a conserved motif that confers Asi1-mediated control when fused to an unrelated DNA-binding protein. Our results indicate that latent precursor forms of Stp1 and Stp2 inefficiently enter the nucleus; however, once there, Asi1 restricts them from binding SPS sensor–regulated promoters. These findings reveal an unanticipated role of inner nuclear membrane proteins in controlling gene expression

Regulation of the ubiquitin-proteasome system: characterization of viral and cellular stabilization signals

The ubiquitin-proteasome system plays a fundamental role in virtually every cellular process. Degradation of endogenous proteins by this system is the major source for antigenic peptides that are presented to MHC class I-restricted cytotoxic T cells. The, Epstein-Barr virus (EBV) nuclear antigen-1 (EBNA1) contains a long glycine-alanine (GA) repeat that inhibits proteasomal processing, resulting in a blockage of antigen presentation. The GA repeat acts in cis and can be functionally transferred to other proteins. The aim of the work described in this thesis was to characterize the protective effect of this viral stabilization signal and to identify similar cellular stabilization signals. To evaluate the inhibitory activity of the GA repeat, we used green fluorescent protein (GFP)-based reporters that were targeted for ubiquitin/proteasome-dependent proteolysis by various degrons. Introducing GA repeats of increasing length resulted in enhanced protection of the fluorescent reporter from proteolysis. When provided with a strong degradation signal even EBNA1 could be efficiently degraded. This study showed that a balance between the strength of the degradation signal and the length of the repeat determines the GA repeat-dependent stabilization effect. Next, we tested the ability of the GA repeat to prevent degradation of the tumor suppressor p53 because inactivation of p53 by accelerated degradation is a common event in tumor development. P53-GA repeat chimeras were protected from degradation and showed improved growth inhibitory activity in tumor cells with impaired endogenous p53 activity, suggesting that insertion of the GA repeat could provide a convenient strategy for the stabilization of potential therapeutic proteins. We used the aforementioned GFP reporters to test the protective effect of the GA repeat in the yeast Saccharomyces cerevisiae. Expression of proteins carrying GA repeats required the generation of yeast codon-optimized recombinant GA (rGA) repeats. We found that introduction of rGA repeats in the GFP substrates resulted in stabilization of the proteins in mammalian and yeast cells, indicating that the protective signal targets a conserved mechanism in the ubiquitin-proteasome system. The yeast DNA-repair protein Rad23 is long-lived despite the fact that it is ubiquitinated and interacts with the proteasome. We investigated whether Rad23 contains domains that can protect it from proteasomal degradation. Disruption of the UBA2 domain converted Rad23 into a short-lived protein that is targeted for proteasomal degradation through its ubiquitin-like domain. Insertion of the UBA2 domain from Rad23 or its human homologue HHR23A prevented the degradation of destabilized GFP reporters without causing a general inhibition of proteolysis. We suggest that the Rad23 UBA2 domain functions as a novel cis-acting stabilization signal that confers protection against proteasomal degradation

Regulation of transcription factor latency by receptor-activated proteolysis

The transcription factor Stp1 is endoproteolytically processed in response to extracellular amino acids by the plasma membrane SPS (Ssy1–Ptr3–Ssy5)-sensor. Processed Stp1, lacking a cytoplasmic retention motif, enters the nucleus and induces amino acid transporter gene expression. The SPS-sensor component Ssy5 is a chymotrypsin-like protease with a Pro-domain and a catalytic domain. The Pro-domain, required for protease maturation, is autolytically cleaved from the catalytic domain but remains associated, forming an inactive protease complex that binds Stp1. Stp1 is processed only after amino acid-induced signals cause the dissociation of the inhibitory Pro-domain. Our findings demonstrate that gene expression can be controlled by regulating the enzymatic activity of an intracellular endoprotease

The inner nuclear envelope as a transcription factor resting place

Just as people head to the beaches for a well-deserved rest, accumulating evidence suggests that transcription factors take similar ‘vacations' at the nuclear envelope. Recent studies indicate that the periphery of the nucleus provides a platform for sequestering transcription factors away from chromatin. Several transcriptional regulators, operating in different signal-transduction pathways, have been found to interact physically with components of the inner nuclear membrane. In general, this association seems to restrict access to their target genes and limit their transactivation or transrepression abilities. The mechanisms of inner nuclear membrane association are diverse, and include regulated associations with the nuclear lamina and integral membrane proteins. Together, these findings indicate that the inside of the nuclear envelope functions as a resting place for transcription factors and suggest a more direct role for the nuclear envelope in gene regulation than previously anticipated

The Prodomain of Ssy5 Protease Controls Receptor-Activated Proteolysis of Transcription Factor Stp1 ▿

Extracellular amino acids induce the yeast SPS sensor to endoproteolytically cleave transcription factors Stp1 and Stp2 in a process termed receptor-activated proteolysis (RAP). Ssy5, the activating endoprotease, is synthesized with a large N-terminal prodomain and a C-terminal chymotrypsin-like catalytic (Cat) domain. During biogenesis, Ssy5 cleaves itself and the prodomain and Cat domain remain associated, forming an inactive primed protease. Here we show that the prodomain is a potent inhibitor of Cat domain activity and that its inactivation is a requisite for RAP. Accordingly, amino acid-induced signals trigger proteasome-dependent degradation of the prodomain. A mutation that stabilizes the prodomain prevents Stp1 processing, whereas destabilizing mutations lead to constitutive RAP-independent Stp1 processing. We fused a conditional degron to the prodomain to synthetically reprogram the amino acid-responsive SPS signaling pathway, placing it under temperature control. Our results define a regulatory mechanism that is novel for eukaryotic proteases functioning within cells