Inner Nuclear Membrane Proteins Asi1, Asi2, and Asi3 Function in Concert to Maintain the Latent Properties of Transcription Factors Stp1 and Stp2

Abstract

In yeast the homologous transcription factors Stp1 and Stp2 are synthesized as latent cytoplasmic precursors with N-terminal regulatory domains. In response to extracellular amino acids the regulatory domains are endoproteolytically excised by the plasma membrane-localized SPS sensor. The processed forms of Stp1 and Stp2 efficiently enter the nucleus and induce expression of amino acid permease genes. We recently reported that the inner nuclear membrane protein Asi1 is required to prevent unprocessed forms of Stp1 and Stp2, which ectopically enter the nucleus, from binding SPS sensor-regulated promoters. Here we show that Asi3, an Asi1 homolog, and Asi2 are integral proteins of the inner nuclear membrane that function in concert with Asi1. In cells lacking any of the three Asi proteins, unprocessed full-length forms of Stp1 and Stp2 constitutively induce SPS sensor-regulated genes. Our results demonstrate that the Asi proteins ensure the fidelity of SPS sensor signaling by maintaining the dormant, or repressed state, of gene expression in the absence of inducing signals. This study documents additional components of a novel mechanism controlling transcription in eukaryotic cells.In eukaryotes the nucleoplasm and cytoplasm are separated by the nuclear envelope. The nuclear envelope consists of two closely aligned bilayers, the inner and outer membranes, each with a unique set of resident proteins. The two nuclear membranes are joined at nuclear pore complexes, which function as channels that provide selective entry and exit routes across the nuclear envelope. Aside from rather detailed knowledge regarding the structure of nuclear pores, there is markedly little known regarding non-pore nuclear proteins (1). However, accumulating data indicate that nuclear envelope proteins participate in a variety of important processes including maintenance of nuclear architecture, chromatin organization, signaling, and gene expression (1-3). Significantly, mutations in genes encoding nuclear envelope proteins are linked to at least 15 inherited human diseases and syndromes (4, 5).In mammalian cells, nuclear lamins are involved in an extensive network of protein-protein interactions, including inner nuclear membrane proteins and transcriptional regulators, some of which bind DNA (3, 6). Although yeast cells lack lamin homologs (7), processes associated with the inner nuclear membrane have been shown to influence patterns of gene expression. For example, although silencing is not obligatorily linked to perinuclear anchoring (8), recruitment of chromatin to the nuclear periphery can facilitate gene repression (9). Evidence obtained using genome-wide approaches suggest that transcriptionally active regions of chromosomes localize to nuclear pore complexes (10, 11). Consistently, a number of actively expressed genes have been found to interact with the nucleoporin Nup2; the interactions occur at the promoter region of genes and appear to correlate with early events of gene expression (12, 13). Together findings in yeast and metazoan...

Cdc48 and Ubx1 participate in a pathway associated with the inner nuclear membrane that governs Asi1 degradation

“…To obtain mechanistic insight into the quality control systems operating in association with the INM, we have investigated the turnover of three native well-characterized and fully functional INM proteins as paradigmatic INMAD substrates -i.e. Asi1, Asi2 and Asi3 (Boban et al, 2006;Forsberg et al, 2001;Zargari et al, 2007). The Asi proteins exhibit distinct turnover rates -Asi1 t 1/2 ≤30 min, Asi2 t 1/2 ≈45 min and Asi3 t 1/2 >90 min (Fig.…”

“…The Asi proteins have been identified as negative regulators of the SPS sensing pathway that are required to suppress gene expression in the absence of amino-acid-mediated induction (Boban et al, 2006;Forsberg et al, 2001;Zargari et al, 2007). We tested the possibility that Asi1 becomes targeted for downregulation in an SPS-sensor-dependent manner.…”

“…Asi1 and Asi3 are homologous proteins: Asi1 (624 residues) and Asi3 (669 residues) are 31% identical and 42% similar in their sequences (Boban et al, 2006;Forsberg et al, 2001;Zargari et al, 2007). Both proteins have two equally sized domains -an N-terminal domain with five hydrophobic segments and a nucleoplasmically oriented hydrophilic C-terminal domain with a conserved Zn 2+ -binding RING motif.…”

Differential Regulation of Transcription Factors Stp1 and Stp2 in the Ssy1-Ptr3-Ssy5 Amino Acid Sensing Pathway

“…It is likely that Grr1 may also play a negative regulatory role by maintaining a low level of fulllength Stp1 in the cytoplasm. Moreover, full-length Stp1 and Stp2 have been reported to "leak" into the nucleus in asi1/ 2/3⌬ mutant cells and activate target gene expression (30,40,42). Grr1 is the primary F-box protein required for degradation of full-length Stp1 in the cytoplasm, whereas both Cdc4 and Grr1 contribute to degradation of processed Stp1, which is mostly in the nucleus.…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…The transcription factors Stp1 and Stp2 (DNAbinding motifs, green boxes) are synthesized as inactive precursors that localize to the cytosol due to the presence of their N-terminal regulatory domain (anchor) that prevents them from efficiently entering the nucleus. Low levels of full-length Stp1 and Stp2 that escape cytoplasmic retention (dashed arrow, left panel) are prevented from derepressing AAP gene expression due to activity of the Asi complex (Asi1-Asi2-Asi3) (Boban et al 2006;Zargari et al 2007). In the presence of extracellular amino acids (right panel), the SPS (Ssy1-Ptr3-Ssy5) sensor activates the intrinsic proteolytic activity of the Ssy5 protease, resulting in the endoproteolytic processing of Stp1 and Stp2 (scissors).…”