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1.
Fig 6

Fig 6. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Dependence of Rim13 localization on the C-terminal region. Strains RIM13-GFP vps4Δ, RIM131–650-GFP vps4Δ, or RIM131–700-GFP vps4Δ were grown to log phase and imaged in CSM for localization of the GFP foci. Scale bar, 5 μm.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
2.
Fig 3

Fig 3. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Rim13-GFP localization dependence on Rim20 and Snf7. (A) Log-phase vps4Δ Rim13-GFP, snf7Δ vps4Δ Rim13-GFP, and rim20Δ vps4Δ Rim13-GFP cells grown in YPD were imaged in CSM. Scale bar, 5 μm. (B) The numbers of cells displaying fluorescent foci were quantified.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
3.
Fig 9

Fig 9. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Model for assembly of the proteolytic complex. (A) Under acidic growth conditions, there is little assembly of Rim13 or Rim20 with ESCRT. Rim20 may interact with the Rim101 C-terminal region. (B) When cells encounter alkaline growth conditions, Rim20 first assembles with ESCRT and may recruit Rim101 as well. (C) Once Rim20-ESCRT is assembled, Rim13 is recruited. Rim13 then cleaves Rim101 to release the N-terminal region, which is the active transcription factor.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
4.
Fig 1

Fig 1. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

pH-dependent localization of Rim13. Log-phase Rim13-GFP cells grown in YPD were shifted to CSM plus 0.1 M HEPES adjusted to pH 4.0 (A and C) or pH 8.3 (B and D) and imaged for appearance of GFP fluorescence immediately after the pH shift. Strains of genotype VPS4 (A and B) and vps4Δ (C and D) were used. We note that vps4Δ strains produced slightly enlarged cells under these growth conditions. Scale bars, 5 μm.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
5.
Fig 8

Fig 8. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Functional activity of Rim13 C-terminal mutants. For gene expression assays, rim13Δ RIM20-GFP cells transformed with pRS316 (empty vector), RIM13-GFP in pRS316, RIM13-LF706AA-GFP, or RIM13-VG708AA-GFP were grown to log phase, harvested, and processed for RNA extraction and cDNA preparation. Quantitative real-time PCR on Rim101-repressed genes NRG1, SMP1, and YPL277c was performed; RIM101 served as a negative control, and TDH3 was the normalization control. Asterisks (*) indicate statistically significant differences compared to the transformant carrying RIM13-GFP.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
6.
Fig 4

Fig 4. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Control of Rim13 localization by Rim101 function. (A) RIM13-GFP cells were compared to rim101Δ RIM13-GFP cells at 10 min after a shift from YPD to CSM plus 0.1 M HEPES (pH 8.3) through fluorescence microscopy. (B) The number of cells displaying fluorescent foci after the shift in panel A was quantified. (C) Complementation analysis was performed by comparing rim101Δ RIM13-GFP cells carrying an empty vector or a RIM101-511 construct at 10 min after a shift from YPD to CSM plus 0.1 M HEPES (pH 8.3) by fluorescence microscopy. (D) The number of cells displaying fluorescent foci after the shift in panel C was quantified. Scale bar, 5 μm.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
7.
Fig 2

Fig 2. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Kinetics and coincidence of Rim13 and Rim20 localization. Log-phase cells expressing Rim13-GFP (A) or Rim20-GFP (B) grown in YPD were shifted to CSM plus 0.1 M HEPES adjusted to pH 8.3 and then imaged at the indicated times after pH shift on three independent days. The number of cells displaying fluorescent foci after the shift was quantified and plotted. (C to F) A RIM20-GFP vps4Δ strain transformed with pRS315 plasmid bearing RIM13-tdTomato was imaged in CSM for colocalization of GFP and tdTomato fluorescence. For a representative field of cells, the signals from GFP (C), tdTomato (D), and both GFP and tdTomato (E) are shown. Scale bar, 5 μm. The rectangles in panels C and D illustrate regions that were scanned for signal intensity. (F) Signal intensities of GFP (green line) and tdTomato (red line) across select regions are graphed. The four panels display the scans across four fields, and the left-hand panel is the scan of the region indicated by the rectangles in panels C and D.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
8.
Fig 5

Fig 5. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Functional role of the Rim13 C-terminal region. A rim13Δ strain carrying RIM13 truncation derivatives, each with a C-terminal 3×FLAG tag, was assayed for Rim13 expression and function. (A) A diagram depicts the C-terminal truncation derivatives. (B) Cells carrying each RIM13 truncation were transformed with a URA3-V5-RIM101 processing reporter plasmid (2), were grown overnight in YPD, diluted and grown to log phase, harvested, lysed, and assayed by immunoblotting with anti-V5 antibody. (C) Anti-FLAG antibody immunoblots were performed on the same extracts as in panel B to detect Rim13 derivatives. The asterisks indicate background proteins that are independent of any introduced FLAG tag. (D) Growth of serial dilutions of each strain on YPD plates with 1 M NaCl was used to determine RIM13 function. In panels B, C, and D, the samples are displayed as follows: lane 1, WT; lane 2, rim13Δ; lane 3, RIM131-450-3×FLAG; lane 4, RIM131-500-3×FLAG; lane 5, RIM131-550-3×FLAG; lane 6, RIM131-600-3×FLAG; lane 7, RIM131-650-3×FLAG; lane 8, RIM131-700-3×FLAG; and lane 9, full-length RIM13-3×FLAG.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.
9.
Fig 7

Fig 7. From: cis- and trans-Acting Localization Determinants of pH Response Regulator Rim13 in Saccharomyces cerevisiae.

Properties of Rim13 alanine scanning mutants. (A) C-terminal predicted sequence of S. cerevisiae Rim13. The symbols beneath each residue refer to sequence similarity among Saccharomyces spp. Rim13 orthologs (asterisks [*] indicate identical amino acids, colons [:] indicate amino acids that have strong similarity, and periods [.] indicate amino acids that have weak similarity). (B) Cells with punctate GFP localization were quantified in transformants of a vps4Δ strain transformed with pRS316 carrying no insert (empty vector) or carrying RIM13-GFP (wild type), RIM13-QL700AA-GFP, RIM13-DE702AA-GFP, RIM13-LE704AA-GFP, RIM13-LF706AA-GFP, RIM13-VG708AA-GFP, RIM13-SS710AA-GFP, RIM13-QK712AA-GFP, RIM13-IR714AA-GFP, RIM13-IE716AA-GFP, RIM13-KY718AA-GFP, RIM13-SD720AA-GFP, RIM13-DV722AA-GFP, RIM13-IP724AA-GFP, and RIM13-K726A-GFP, as indicated from left to right. In the graph, asterisks (*) indicate statistically significant differences, with a P value of <0.005. (C) Expression and stability of GFP fusion constructs was detected by SDS-PAGE and immunoblots of whole-cell lysates of the above strains. Note that there is a background band (as seen in the vector control lane) that cross-reacts with the anti-GFP N-terminal antibody.

Shoba Subramanian, et al. Eukaryot Cell. 2012 October;11(10):1201-1209.

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