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Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

Rogers JV, Rose MD - G3 (Bethesda) (2014)

Bottom Line: Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment.We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p.Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014.

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Prm3p induces Kar5p aggregation along the nuclear envelope. (A) Representative examples of Kar5-TM3-GFP (pMR6364) in wild type (WT; MS7729), prm3Δ (MS7884), kar2-1 (MS8041), and kar8Δ (MS8087) shmoos. (B) Representative examples of pADH1-Kar5-TM3-GFP (pMR6371) in WT and prm3Δ mitotic cells and shmoos. (C) Representative examples of pADH1-Kar5-TM3-GFP and pADH1-mCherry-Prm3 (pMR6433) in MS7670, either separately or coexpressed. GFP images within each panel are displayed at the same brightness. Scale bars, 2 μm. GFP, green fluorescent protein.
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fig4: Prm3p induces Kar5p aggregation along the nuclear envelope. (A) Representative examples of Kar5-TM3-GFP (pMR6364) in wild type (WT; MS7729), prm3Δ (MS7884), kar2-1 (MS8041), and kar8Δ (MS8087) shmoos. (B) Representative examples of pADH1-Kar5-TM3-GFP (pMR6371) in WT and prm3Δ mitotic cells and shmoos. (C) Representative examples of pADH1-Kar5-TM3-GFP and pADH1-mCherry-Prm3 (pMR6433) in MS7670, either separately or coexpressed. GFP images within each panel are displayed at the same brightness. Scale bars, 2 μm. GFP, green fluorescent protein.

Mentions: To further understand how Kar5p might interact with other nuclear fusion proteins, we expressed Kar5-TM3-GFP in nuclear fusion mutants prm3Δ, kar2-1, and kar8Δ. In kar2-1 and kar8Δ shmoos, Kar5-TM3-GFP appeared normal; it enriched strongly at the SPB, and formed several bright puncta elsewhere on the nuclear envelope (Figure 4A). In contrast, in prm3Δ shmoos Kar5-TM3-GFP enriched strongly at the SPB, but did not form additional puncta, and instead was diffusely localized along the nuclear envelope and peripheral ER (Figure 4A). This suggested that Prm3p might have a role in restricting Kar5p to the nuclear envelope and causing it to aggregate.


Kar5p is required for multiple functions in both inner and outer nuclear envelope fusion in Saccharomyces cerevisiae.

Rogers JV, Rose MD - G3 (Bethesda) (2014)

Prm3p induces Kar5p aggregation along the nuclear envelope. (A) Representative examples of Kar5-TM3-GFP (pMR6364) in wild type (WT; MS7729), prm3Δ (MS7884), kar2-1 (MS8041), and kar8Δ (MS8087) shmoos. (B) Representative examples of pADH1-Kar5-TM3-GFP (pMR6371) in WT and prm3Δ mitotic cells and shmoos. (C) Representative examples of pADH1-Kar5-TM3-GFP and pADH1-mCherry-Prm3 (pMR6433) in MS7670, either separately or coexpressed. GFP images within each panel are displayed at the same brightness. Scale bars, 2 μm. GFP, green fluorescent protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4291462&req=5

fig4: Prm3p induces Kar5p aggregation along the nuclear envelope. (A) Representative examples of Kar5-TM3-GFP (pMR6364) in wild type (WT; MS7729), prm3Δ (MS7884), kar2-1 (MS8041), and kar8Δ (MS8087) shmoos. (B) Representative examples of pADH1-Kar5-TM3-GFP (pMR6371) in WT and prm3Δ mitotic cells and shmoos. (C) Representative examples of pADH1-Kar5-TM3-GFP and pADH1-mCherry-Prm3 (pMR6433) in MS7670, either separately or coexpressed. GFP images within each panel are displayed at the same brightness. Scale bars, 2 μm. GFP, green fluorescent protein.
Mentions: To further understand how Kar5p might interact with other nuclear fusion proteins, we expressed Kar5-TM3-GFP in nuclear fusion mutants prm3Δ, kar2-1, and kar8Δ. In kar2-1 and kar8Δ shmoos, Kar5-TM3-GFP appeared normal; it enriched strongly at the SPB, and formed several bright puncta elsewhere on the nuclear envelope (Figure 4A). In contrast, in prm3Δ shmoos Kar5-TM3-GFP enriched strongly at the SPB, but did not form additional puncta, and instead was diffusely localized along the nuclear envelope and peripheral ER (Figure 4A). This suggested that Prm3p might have a role in restricting Kar5p to the nuclear envelope and causing it to aggregate.

Bottom Line: Several kar5 mutant proteins localized properly but did not mediate Prm3p recruitment; other kar5 mutant proteins localized and recruited Prm3p but were nevertheless defective for nuclear fusion, demonstrating additional functions beyond Prm3p recruitment.We identified one Kar5p domain required for SPB localization, which is dependent on the half-bridge protein Mps3p.Finally, a split-GFP assay demonstrated that Kar5p localizes to both the inner and outer nuclear envelope.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544-1014.

Show MeSH
Related in: MedlinePlus