Abstract
SUG1 is an integral component of the 26 S proteasome. Belonging to a novel putative ATPase family, it shares four conserved motifs characteristic of ATP-dependent DNA/RNA helicases. Recombinant rat SUG1 (rSUG1) produced in Escherichia coli was highly purified and characterized in terms of its biochemical properties. The rSUG1 exhibited a Mg2+-dependent ATPase activity. The Km for ATP and Vmax of rSUG1 were 35 microM and 7 pmol of ATP/min/microg of protein, respectively. Both ATPase activity to release [32P]monophosphate and [32P]ATP-labeling activity were coordinately affected by cold ATP severely, GTP and UTP moderately, and CTP little. Interestingly, the rSUG1 ATPase activity was stimulated by poly(U) and poly(C), but not by poly(A), poly(G), or by any forms of DNAs tested. A UV cross-linking assay also indicated poly(U)- and poly(C)-stimulated labeling of rSUG1 with [alpha-32P]ATP. Moreover, the ATPase activity was facilitated by cellular poly(A)+ 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.
Highlights
The 26 S proteasome is a huge protease complex that degrades short-lived proteins related to metabolic regulation and cell cycle progression [1, 2]
We found that rSUG11 exhibited ATPase activity that was stimulated by particular RNA molecules
Expression and Purification of Recombinant Rat SUG1—We cloned rat SUG1 cDNA and found it to encode a protein of 406 amino acids [6] (Fig. 1) and to have exactly the same structure as human p45 [21], the human homolog of yeast SUG1
Summary
Expression and Purification of Rat SUG1—SUG1 cDNA was cloned from a rat liver cDNA library as described previously [6]. 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 was gradually removed by dialysis. These proteins were analyzed by 10% SDS-PAGE and stained with Coomassie Brilliant Blue. Reactions (20 l) contained 0.5 g of purified rSUG1 in buffer A (20 mM Tris-HCl (pH 7.5), 70 mM KCl, 2.5 mM MgCl2, 1.5 mM dithiothreitol, 500 M ATP, and 1.25 Ci of [␥-32P]ATP). Preparation of Cellular RNAs—Cellular total RNA was prepared from rat liver by use of cesium trifluoroacetate as described [19]. MSS1 is a rat homolog of the human MSS1 and is a part of the 26 S proteasome [6]
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