SUG1, a Component of the 26 S Proteasome, Is an ATPase Stimulated by Specific RNAs

Abstract

RNA, but not by poly(A)؊ RNA. RNA transcribed in vitro from cDNA encoding a b-Zip protein could stimulate the ATPase activity. This is the first report to demonstrate a specific RNA requirement for ATPase with respect to the proteasomal ATPases. Our present work suggests that SUG1 can specifically interact with protein-coding RNA (mRNA) and play some roles in mRNA metabolism.The 26 S proteasome is a huge protease complex that degrades short-lived proteins related to metabolic regulation and cell cycle progression (1, 2). It is composed of the 20 S catalytic core and an ATPase-containing 22 S regulatory complex (3, 4). The human 22 S complex contains at least five highly related putative ATPases, i.e. TBP1, TBP7, S4, MSS1, and p45 (a homolog of yeast SUG1), which are members of a novel ATPase family named the AAA family (ATPases associated with a variety of cellular activities) (5). The family members have a highly conserved ATPase module with 200 amino acids and fulfill a large diversity of functions. It was demonstrated that the five ATPases contain four conserved motifs characteristic of putative ATP-dependent RNA/DNA helicases (6 -8). We previously reported constant distances between each motif among proteasomal ATPases, suggesting the functional importance of these motifs (6).Of the above ATPases, SUG1 (11) has been shown by various studies to be involved in transcriptional regulation in addition to proteolytic function. The yeast SUG1 was reported to be a component of the RNA polymerase II (pol II) holoenzyme (12, 13), and Trip1, a human homolog of the SUG1, interacts with the thyroid hormone receptor (14). Baur et al. (15) also reported that mouse SUG1 interacts with various nuclear receptors. However, it is still controversial as to whether or not SUG1 acts as an intrinsic transcription factor because one cannot eliminate the possibility that those ATPases are involved in degradation of transcription factors mediated by the 26 S proteasome. Actually, some transcription factors are regulated by proteasome-dependent proteolysis (1). Biochemical study is required to resolve issues such as how the ATPases are involved in RNA metabolism such as transcriptional regulation. In this study, we report the purification and characterization of rat SUG1. We found that rSUG1 1 exhibited ATPase activity that was specifically stimulated by particular RNA molecules. EXPERIMENTAL PROCEDURESExpression and Purification of Rat SUG1-SUG1 cDNA was cloned from a rat liver cDNA library as described previously (6). Histidinetagged rSUG1 was overexpressed in Escherichia coli by use of the pET vector system (16). Insoluble recombinant rSUG1 was purified by Ni 2ϩ -agarose under denaturing conditions according to the instructions supplied by Qiagen. The resulting proteins were further subjected to a preparative SDS-PAGE and recovered from the excised gels. The protein was redissolved in a urea-containing buffer (25 mM Tris-HCl (pH 7.5), 0.3 M NaCl, 1 mM 2-mercaptoethanol, 0.1% Nonidet P-40, 10% glycerol, and 8 M urea), and the urea w...

Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA‐binding protein and a novel transcriptional activator, TIP120

“…Yeast and mouse SUG1s have been reported to bind to its cognate TBP. In a previous work, we determined that rat SUG1 was 44 kDa in molecular mass (Makino et al 1997). Taken together, we conclude that TIP43 contains SUG1.…”

“…HIV Tat has been found to stimulate transcription elongation mediated by a cellular factor (Zhou & Sharp 1995), and MSS1 and TBP1 are thought to regulate HIV gene expression via Tat. We demonstrated that rat SUG1 ATPase was stimulated by specific RNAs (Makino et al 1997). Recently, Russell et al (1999) reported that the yeast SUG1 and SUG2 are involved in nucleotide-excision repair.…”

The Nuclear Vitamin D Receptor: Biological and Molecular Regulatory Properties Revealed

“…4 for simplicity) likely targets the receptor for ubiquitination and subsequent proteolysis by the 26S proteasome complex. (185)(186)(187) The availability of a cell-free transcription system in which VDR-RXR heterodimers activate transcription in a 1,25(OH) 2 D 3 -dependent fashion (188) should facilitate studies of transcriptional control by VDR, such as demonstrating direct effects by coactivators, promoter-specific factors, or corepressors. Ultimately, with such cell-free transcription systems it may also be feasible to incorporate elements of chromatin structure such as the aforementioned histones, in order to develop a system that truly mimics VDR action in vivo while at the same time permitting manipulations, in vitro.…”

The Ubiquitin-Conjugating Enzyme UBCH7 Acts as a Coactivator for Steroid Hormone Receptors

“…It has been suggested that the ubiquitin-proteasome pathway plays an important regulatory role in nuclear receptor function, adding a new dimension to the field of nuclear hormone receptors (10,32,33,39). Besides ubiquitin-protein ligases, such as E6-AP; RSP5 (reverses Spt phenotypes) and its human homologue, RPF-1 (receptor potentiation factor 1) (23,39); UBA3, a ubiquitin-activating enzyme of the NEDD8 pathway (12,16); the GAL4 suppressor SUG1 (suppressor of Gal4D lesions 1) (14,33); and UBC9 (46), the human homologue of Saccharomyces cerevisiae E2 ubiquitin-conjugating enzyme of the sumoylation pathway, are some of the components of the ubiquitin-proteasome pathway and ubiquitin-like pathways that have been shown to modulate the properties of steroid receptors.…”