Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae.

Abstract

A putative Z‐DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z‐DNA binding assay using [32P]poly(dG‐m5dC) and [32P]oligo(dG‐Br5dC)22 in the presence of B‐DNA competitor. Poly(dG‐Br5dC) in the Z‐form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG‐dC) and poly(dA‐dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG‐dC)7 segment in the Z‐form was an excellent competitor. A Southwestern blot using [32P]poly(dG‐m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N‐terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z‐DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

Heat‐shock proteins as molecular chaperones

“…Although no proteins similar to GrpE have yet been reported in eucaryotic cells, five DnaJ-like proteins have been discovered in yeast; YDJl/MASS (Atencio and Yaffe, 1992;Caplan and Douglas, 1991), SZSl (Luke et al, 1991), SCJl (Blumberg and Silver, 1991), SEC63/NPLl (Sadler et al, 1989) and Zuotin (Zhang et al, 1992). In addition two dnaJ homologues have been cloned from human cells (Cheetham et al, 1992;Raabe and Manley, 1991).…”

Mdj1p, a novel chaperone of the DnaJ family, is involved in mitochondrial biogenesis and protein folding

“…For Sec63p and Zuolp only, the J region is shown (residues 125-199 and 97-171, respectively) because only this region shows similarity to the other members of the protein family. DnaJ (Bardwell et al, 1986); SEC63 (Rothblatt et al, 1989); YDJl (Caplan and Douglas, 1991); SISI (Luke et al, 1991);SCJl (Slumberg and Silver, 1991); and ZUOl (Zhang et al, 1992). growth defect was relieved by transformation with a plasmid (pMDJ315) containing the complete MDJ7 coding and regulatory sequences.…”

“…In the yeast Saccharomyces cerevisiae, several genes of DnaJ homologs have so far been characterized: SfC63, ScJl, YWl,SISl,ZUOl, andXW1 (Rothblattetal., 1989;Sadler et al, 1989;Blumberg and Silver, 1991;Caplan and Douglas, 1991;Atencio and Yaffe, 1992;Luke et al, 1991;Zhang et al, 1992;Schwarz et al, in press). Among these, Sec63p and Ydjl p have been shown to be involved in protein translocation across intracellular membranes (Rothblatt et al, 1989;Caplan et al, 1992).…”

Hydrogel scaffolds composed of genetically synthesized self-assembling peptides for three-dimensional cell culture

“…One such self-assembling peptide, named RADA16 (RADARADAR-ADARADA), is a designed oligopeptide whose sequence is based on EAK16 (AEAEAKAKAEAEAKAK), which was originally identified as a set of tandem repeats in the yeast protein Zuotin. 14,15 To design RADA16, the lysine and glutamic acid residues of EAK16 were altered to arginine and aspartic acid residues, respectively, and the sequence order was modulated. The resulting RADA16 peptide is composed of four repeating units of the RADA amino-acid sequence and forms a stable b-sheet structure.…”