A RIM101-dependent operator element within NRE^DIT. (A) The sequence of the 76-bp NRE^DIT, from nt −537 to −462 upstream of the DIT1 gene, is given in the top line and denoted as NRE76. The PacC^DIT sequence is boxed and annotated to show the A-to-T mutation at nt −480. The sequences spanned by NRE53, NRE44, and NRE30 are also shown. (B) Strains W303-1A (wild type, open bars), Y108 (frd5-1, gray bars), and Y104 (rim101Δ, black bars) were separately transformed with pLG312, pLG+NRE76, pLG+NRE53, and pLG+NRE30(+). Repression (n-fold) of gene expression is given as the ratio of β-galactosidase activity measured in crude extracts of cells in which the plasmid-borne CYC1-lacZ reporter gene had no insert to the activity measured in cells from the same strain in which the plasmid-borne CYC1-lacZ reporter gene contained the indicated NRE site inserted between the UAS^CYC1 and the TATA box (see Materials and Methods). (C) Strains W303-1A (wild type [WT]), Y104 (rim101Δ), and Y170 (tup1Δ) were separately transformed with each of the plasmids indicated in the first column and pLG312. The arrowheads in the schematic of the inserts given in the second column denote the orientation of the inserts in the plasmid-borne CYC1-lacZ reporter genes relative to the DIT1 gene. NRE30m and 3×NRE30m are identical to NRE30(−) and 3×NRE30, respectively, with the exception that the A residue at nt −480 has been mutated to a T residue and the orientation of the inserts is reversed in 3×NRE30m relative to 3×NRE30. Repression (n-fold) given in the last three columns refers to the ratio of β-galactosidase activity measured in cells containing pLG312 to the activity measured in cells of the same strain containing the plasmid-borne CYC1-lacZ reporter with the indicated insert. The activities reported in this figure and in Fig. are the average activities obtained from three or more independent cultures analyzed at the same time. Each experiment was repeated from one to five times; although absolute values varied between experiments, the relative levels of β-galactosidase activities were similar.

A RIM101-independent operator site within the bipartite NRE^DIT. (A) The sequence of NRE76 is given in the top line with the PacC^DIT site boxed, and the four base-pair mutations present in NRE42m are indicated by arrows. The sequences spanned by NRE42 (nt −505 to nt −464), NRE25 (nt −505 to nt −481), and NRE22D (nt −486 to nt −464) are shown. (B) Comparison of expression levels of plasmid-borne CYC1-NRE-lacZ reporter genes in wild-type cells. Repression (n-fold) of gene expression was measured as the ratio of β-galactosidase activity in cells in which the plasmid-borne CYC1-lacZ reporter gene had no insert to the activity in cells from the same strain in which the plasmid-borne CYC1-lacZ reporter gene contained NRE76, 1×NRE25, 2×NRE25, 3×NRE25, 1×NRE22D, 2×NRE22D, or 3×NRE22D inserted between the UAS^CYC1 and the TATA box. Relative repression is the ratio of the repression (fold) mediated by the indicated insert normalized to the repression (fold) mediated by NRE76. (C) Comparison of expression levels of plasmid-borne CYC1-NRE-lacZ reporter genes in wild-type (W303-1A; open bars), rim101Δ (Y104; gray bars), and tup1Δ (Y170; black bars) cells. Relative repression values were obtained as described above for panel B. (D) Introduction of mutations upstream of the PacC^DIT site reduces NRE42-mediated repression in wild-type and rim101Δ cells. Colonies derived from wild-type cells and rim101Δ cells containing pLG312 (no insert), pLG+NRE76, pLGn+NRE42, pLGn+3×NRE25, pLGn+3×NRE22D, pLGn+NRE42m, or pLGΔSS (no UAS) were overlaid with X-Gal-containing agar (upper left panel). The image is a scan of the colonies after incubation at 30°C. The table gives units of β-galactosidase activity in wild-type (WT) and rim101Δ cells containing pLG312 (no insert), pLGn+NRE42, pLGn+NRE42m-1, or pLGΔSS (no UAS). Relative repression values calculated from these data as described above are given in the bar graph.

Rim101 binds to the PacC^DIT site in vitro and in vivo. (A) A truncated version of Rim101 extending from residue 1 to residue 289 was synthesized in vitro and tested for its ability to bind to a radiolabeled NRE22D-containing oligonucleotide by EMSA as described in Materials and Methods. An equivalent amount of Rim101(1-289) protein was incubated with a radiolabeled NRE22D-containing double-stranded oligonucleotide (5′-GATCCTTGCCAAGAAAAAATAAAAAGGATC-3′ [the PacC^DIT site is shown in boldface type]) in the absence of competitor DNA (lane 2) and in the presence of a 10-, 50-, 100-, or 250-fold excess of nonlabeled NRE22D-containing double-stranded oligonucleotide (lanes 3 to 6) or a nonlabeled mutant version of this double-stranded oligonucleotide (lanes 7 to 10) as specific and nonspecific competitors, respectively. The mutant oligonucleotide had a 1-bp change within the PacC^DIT site, changing the sequence from 5′-TGCCAAGA-3′ to 5′-TGCCTAGA-3′. The reaction of lane 1 contained only the probe DNA. WT, wild type. (B) A Gal4^AD-Rim101(1-289) fusion protein expressed in vivo activates an NRE22D-containing reporter gene. W303-1B was transformed with pACTII-Rim101(1-289) (Gal4^AD-Rim101^DB) or pACTII (Gal4^AD), as indicated at the top of the panel. As indicated on the right of the panel, these transformants were then transformed with pLG312 (no insert); pLGΔSS (no UAS); pLGΔSS+4×NRE22D, which contains a 4×NRE22D-lacZ gene reporter gene; pLGΔSS+1×NRE22D, which contains a 1×NRE22D-lacZ reporter gene; or pLGΔSS+2×NRE25, which contains a 2×NRE25-lacZ reporter gene. Colonies representing two distinct transformants of each strain were grown up and overlaid with X-Gal-containing agar to monitor β-galactosidase expression.

Rim101-dependent, 3×NRE22D-mediated repression requires the RIM signaling pathway. (A) The wild-type (WT) strain or the indicated rim deletion strains from the yeast deletion collection, as denoted on the left-hand side of the panel, were transformed with pLGn+3×NRE22D, and β-galactosidase expression was monitored by a colony overlay assay. (B) Expression of Rim101(1-531)0.3HA restores NRE22D-mediated repression to a rim13 strain. Wild-type, rim101Δ, RIM101(1-531).3HA-HIS3MX6, rim13Δ, and rim13Δ RIM101(1-531)0.3HA-HIS3MX6 strains, as denoted on the right-hand side of the panel, were transformed with pLG312n (no insert) or pLGn+3×NRE22D, and β-galactosidase expression was monitored by a colony overlay assay.

NRG1 is required for 3×NRE25- but not 3×NRE22D-mediated repression; RIM101, DFG16, YGR122w, and VPS36 are required for 3×NR22D- but not 3×NRE25-mediated repression. Colonies of the BY4741-derived deletion strains, as indicated on the right side of the panel, that contained pLGn+NRE42, pLGn+3×NRE25, or pLGn+3×NRE22D, as indicated at the top of the panel, were overlaid with X-Gal-containing agar. WT, wild type.

Formation of Nrg1-NRE25 and Nrg1-Rim101-NRE42 protein-DNA complexes in vitro as assessed by EMSA and interaction of Nrg1 and Rim101 in vitro as assessed by affinity chromatography. (A) Nrg1 binds to NRE25 in vitro. Bacterially expressed MBP-Nrg1 was affinity purified and incubated with a radiolabeled double-stranded oligonucleotide containing NRE25 (5′-CCATAAATAAAAGGGTTCTCTTGCC-3′) prior to electrophoresis on a nondenaturing 6% polyacrylamide gel. The reactions of lanes 4 to 11 contained equivalent amounts of MBP-Nrg1; the reactions of lanes 5 to 7 contained increasing amounts of a nonlabeled NRE25-containing double-stranded oligonucleotide as specific competitor DNA; and the reactions of lanes 8 to 10 contained increasing amounts of a nonlabeled mutant NRE25-containing double-stranded oligonucleotide as a nonspecific competitor (the wild-type sequence 5′-AGGGT-3′ [indicated in boldface type in the sequence given above] was changed to 5′-CTGTA-3′). The reaction of lane 1 contained no protein, and the reaction of lane 11 contained bacterially expressed MBP. The reactions of lanes 2 and 3 contained 5- and 2.5-fold of the amount of MBP-Nrg1 present in the reaction of lane 4. (B) Rim101(1-289) does not interact with the NRE25 site, and MBP-Nrg1 does not interact with the NRE22D site. An EMSA was performed with bacterially expressed Rim101(1-289) present in the soluble fraction of a crude cell lysate (lanes 2 and 4) or affinity-purified bacterially expressed MBP-Nrg1 (lanes 6 and 8) that had been incubated with a radiolabeled oligonucleotide containing NRE22D (Fig. ) (lanes 2 and 8) or NRE25 (lanes 4 and 6). The reactions of lanes 1, 3, 5, and 7 contained probe only. (C) An Nrg1-Rim101-DNA complex forms in vitro with NRE42 but not with NRE42m-2 or NRE42m-1. Bacterially expressed MBP-Nrg1 (lanes 2, 4, 6 to 8, and 12 to 14) or bacterially expressed Rim101(1-289) (lanes 3, 4, and 9 to 14) was incubated with a radiolabeled oligonucleotide containing NRE42 (see Materials and Methods) (lanes 1 to 4), NRE42m-2 (5′-CCATAAATAAAAGGGTTCTCTTGCCTAGAAAAAATAAAAAGGCC-3′ [the mutation is in boldface type]) (lanes 5 to 14, upper panel) or NRE42m-1 (see Materials and Methods) (lanes 5 to 14, lower panel). The reactions of lanes 1 and 5 contained no protein. The reactions of lanes 6 to 8 and lanes 12 to 14 (upper panel) contained increasing amounts of MBP-Nrg1; the reactions of lanes 9 to 11 and lanes 12 to 14 (lower panel) contained increasing amounts of Rim101(1-289). Only the portions of the autoradiograms representing the protein-DNA complexes are shown. (D) Interaction between Nrg1 and Rim101 in vitro. Western blot analysis with HRP-conjugated anti-HA antibodies of samples from an affinity chromatography experiment (see Materials and Methods) is shown. A lysate from induced bacterial cells expressing MBP (lanes 2 to 6) or MBP-Nrg1 (lanes 10 to 14) was incubated with amylose-Sepharose to prepare an affinity resin to capture Rim101 from a yeast cell lysate. The following samples were analyzed: an aliquot of uninduced bacterial cells (U, lanes 1 and 9) and an aliquot of the soluble fraction of a lysate prepared from induced bacterial cells (I, lanes 2 and 10) that contained a vector for expression of MBP (lanes 1 and 2) or MBP-Nrg1 (lanes 9 and 10); an aliquot of a flowthrough fraction after the bacterial lysate-resin (amylose-Sepharose) mixture had been loaded into a column (FT, lanes 3 and 11); an aliquot of a wash fraction from the columns after a lysate prepared from yeast cells expressing Rim101(1-531).3HA had been run through the columns (W, lanes 4 and 12); aliquots of resin-bound protein that had been eluted with maltose (E₁ and E₂, lanes 5, 6, 13, and 14); an aliquot of the lysate prepared from yeast cells expressing Rim101(1-531).3HA (lanes 7 and 15) or Rim101 (lanes 8 and 16). The asterisks on the left denote bacterial proteins that cross-reacted with the anti-HA antibody; the position of Rim101(1-531).3HA is denoted on the right. (E) The filter of panel C was stripped and reprobed with anti-MBP antibodies as the primary antibody and an HRP-conjugated secondary antibody. Lanes 1 through 8 correspond to lanes 2, 3, 5, 6, 10, 11, 13, and 14, respectively, of panel D.

Expression of the CYC1-NRE42-lacZ reporter gene and the (−537)DIT1-lacZ reporter gene is higher in an nrg1 rim101 strain than in either an nrg1 strain or a rim101 strain. (A) Colonies of wild-type (WT) (W303-B), nrg1Δ (KRY302), rim101Δ (KRY308), and nrg1Δ rim101Δ (KRY318) cells and tup1Δ (Y169) cells containing pLG312n, a plasmid with a CYC1-lacZ reporter gene (no insert); pLGn+NRE42, a plasmid with a CYC1-NRE42-lacZ reporter gene; and p(−537)DIT1-lacZ, a plasmid with a (−537)DIT1-lacZ reporter gene, were overlaid with X-Gal-containing agar. (B) The table gives units of β-galactosidase activity measured in extracts from cells containing pLG312 (no insert) or pLGn+NRE42 (NRE42).

Involvement of ESCRT components, Dfg16, and Ygr122w in 3×NRE22D-mediated repression and processing of Rim101. (A) Colonies of the BY4741-derived deletion strains, as indicated on the sides of the panels, containing pLG312n (no insert), pLGn+3×NRE25, or pLGn+ 3×NRE22D, as indicated above the panels, were overlaid with X-Gal-containing agar. I, II, IIIA, and IIIB refer to ESCRT complexes. (B) Repression of the CYC1-3×NRE22D-lacZ reporter gene can be restored in the mutant strains by expression of a truncated version of Rim101. For each mutant strain identified on the left of the panel, the top two rows of colonies were from cells that contained the wild-type (WT) RIM101 gene, and the bottom two rows of colonies were from cells that contained the RIM101(1-531).3HA-HIS3MX6 allele, as indicated on the right. The cells also contained pLG312n (no insert) or pLGn+3×NRE22D, as indicated above the panels. For panels A and B, composite images were prepared from scans of plates that had been overlaid with X-Gal-containing agar after color development. Each plate had control colonies consisting of wild-type cells and rim101Δ cells containing pLG312n or pLG+3×NRE22D to ensure equivalent color development. (C) Western blot of an SDS-polyacrylamide gel containing aliquots of crude lysates prepared from the indicated BY4741-derived deletion strains that expressed a full-length internally HA-epitope-tagged Rim101 protein. The blot was probed with HRP-conjugated anti-HA antibodies.