Cotransport of the Heterodimeric Small Subunit of the Saccharomyces cerevisiae Ribonucleotide Reductase Between the Nucleus and the Cytoplasm

Abstract

Ribonucleotide reductase (RNR) catalyzes the rate-liming step in de novo deoxyribonucleotide biosynthesis and is essential in DNA replication and repair. Cells have evolved complex mechanisms to modulate RNR activity during normal cell cycle progression and in response to genotoxic stress. A recently characterized mode of RNR regulation is DNA damage-induced RNR subunit redistribution. The RNR holoenzyme consists of a large subunit, R1, and a small subunit, R2. The Saccharomyces cerevisiae R2 is an Rnr2:Rnr4 heterodimer. Rnr2 generates a diferric-tyrosyl radical cofactor required for catalysis; Rnr4 facilitates cofactor assembly and stabilizes the resulting holo-heterodimer. Upon DNA damage, Rnr2 and Rnr4 undergo checkpoint-dependent, nucleus-to-cytoplasm redistribution, resulting in colocalization of R1 and R2. Here we present evidence that Rnr2 and Rnr4 are transported between the nucleus and the cytoplasm as one protein complex. Tagging either Rnr2 or Rnr4 with a nuclear export sequence causes cytoplasmic localization of both proteins. Moreover, mutations at the Rnr2:Rnr4 heterodimer interface can affect the localization of both proteins without disrupting the heterodimeric complex. Finally, the relocalization of Rnr4 appears to involve both active export and blockage of nuclear import. Our findings provide new insights into the mechanism of DNA damage-induced RNR subunit redistribution.

Yeast Dun1 Kinase Regulates Ribonucleotide Reductase Small Subunit Localization in Response to Iron Deficiency

“…Protein Analyses-Total protein extracts were obtained by using glass beads disruption in 20% trichloroacetic acid as described (48). Protein extracts from an equal number of cells were separated in SDS-PAGE gels and transferred onto nitrocellulose membranes.…”

Dif1 Controls Subcellular Localization of Ribonucleotide Reductase by Mediating Nuclear Import of the R2 Subunit

“…Two different extraction solutions/buffers were used. For immunoblotting to detect steady-state levels of Dif1 protein, trichloroacetic acid was employed to extract protein from 1 ϫ 10 7 to 1 ϫ 10 8 mid-log-phase cells for each loading (2). For phosphatase treatment, protein extracts were prepared in buffer B (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, supplemented with 1ϫ protease inhibitor cocktail from Roche Applied Science) and centrifuged at 13,400 ϫ g for 15 min to remove debris.…”

Investigation of in Vivo Roles of the C-terminal Tails of the Small Subunit (ββ′) of Saccharomyces cerevisiae Ribonucleotide Reductase

“…Plasmids pMH813 (pRS415-P RNR2 -3ϫMyc-RNR2) and pMH569 (pRS413-P RNR4 -HA-RNR4) contain an N-terminal in-frame triple-Myc and HA epitope between the promoter and coding sequences of RNR2 and RNR4, respectively (25). The rnr2-Y376F and rnr4-Y323F harboring plasmids pMH1669 and pMH1668 were constructed by using site-directed mutagenesis on pMH813 and pMH569 and were introduced into the plasmid shuffle strains MHY593 (MATa, rnr2::KanMX6, pMH881 (URA3CENRNR2)) and MHY20 (rnr4::LEU2, pMH140 (URA3CENRNR4)), respectively, as described (25).…”

“…The rnr2-Y376F and rnr4-Y323F harboring plasmids pMH1669 and pMH1668 were constructed by using site-directed mutagenesis on pMH813 and pMH569 and were introduced into the plasmid shuffle strains MHY593 (MATa, rnr2::KanMX6, pMH881 (URA3CENRNR2)) and MHY20 (rnr4::LEU2, pMH140 (URA3CENRNR4)), respectively, as described (25).…”

“…In contrast with the small ␤ 2 subunit of most organisms, the yeast small subunit is a heterodimer in vivo, ␤␤Ј, encoded by the RNR2 and RNR4 genes, respectively (22)(23)(24)(25). ␤Ј is structurally homologous to ␤ but lacks three iron ligands, and as a consequence, contains no metallo-cofactor (18,22,(25)(26)(27). Although ␤Ј is catalytically inactive, it is required for converting ␤ into a conformation that is competent for iron loading and Y ⅐ formation in vitro and in vivo (23,24,28).…”