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N-terminal acetylation and replicative age affect proteasome localization and cell fitness during aging.

van Deventer S, Menendez-Benito V, van Leeuwen F, Neefjes J - J. Cell. Sci. (2014)

Bottom Line: By using a microscopy-based genome-wide screen, we identified genetic factors involved in these processes.Both relocalization of the proteasome and PSG formation were affected by two of the three N-acetylation complexes.These N-acetylation complexes also had different effects on the longevity of cells, indicating that each N-acetylation complex has different roles in proteasome location and aging.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute and Netherlands Proteomics Center, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

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Proteasome localization in nutrient-starved cells correlates with replicative age. (A) Live-cell microscopy of yeast cells in starvation shows various 20S proteasome localizations as is visualized by endogenous expression of a GFP-tagged β1 subunit (Pre3). Cells were stained with CFW to assess the replicative age of individual cells. (B) β1–GFP localization and Hoechst 33342 staining was used to define four different phenotypes: cells with cytosolic PSGs (PSG), cells with nuclear enrichment of proteasomes (Nuclear), cells that display both a nuclear enrichment of proteasomes and PSGs (Nuclear + PSG), and cells without a clear enrichment of proteasomes in PSGs or nuclei (Equal). (C) Based on CFW staining of bud scars, three different replicative age groups were defined: virgin daughter cells without bud scars (V); young mother cells with 1–2 bud scars (YM); and old mother cells with more than two bud scars (OM). (D) The prevalence of the different proteasome phenotypes in living cells from each age group was calculated by dividing the number of cells with a certain phenotype in a particular age group over the total number of cells in this age group. Results are mean±s.d. based on three independent experiments. Significance was calculated with a paired, two-tailed Student's t-test (*P<0.05, **P<0.01). Scale bars: 5 µm.
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f01: Proteasome localization in nutrient-starved cells correlates with replicative age. (A) Live-cell microscopy of yeast cells in starvation shows various 20S proteasome localizations as is visualized by endogenous expression of a GFP-tagged β1 subunit (Pre3). Cells were stained with CFW to assess the replicative age of individual cells. (B) β1–GFP localization and Hoechst 33342 staining was used to define four different phenotypes: cells with cytosolic PSGs (PSG), cells with nuclear enrichment of proteasomes (Nuclear), cells that display both a nuclear enrichment of proteasomes and PSGs (Nuclear + PSG), and cells without a clear enrichment of proteasomes in PSGs or nuclei (Equal). (C) Based on CFW staining of bud scars, three different replicative age groups were defined: virgin daughter cells without bud scars (V); young mother cells with 1–2 bud scars (YM); and old mother cells with more than two bud scars (OM). (D) The prevalence of the different proteasome phenotypes in living cells from each age group was calculated by dividing the number of cells with a certain phenotype in a particular age group over the total number of cells in this age group. Results are mean±s.d. based on three independent experiments. Significance was calculated with a paired, two-tailed Student's t-test (*P<0.05, **P<0.01). Scale bars: 5 µm.

Mentions: We visualized proteasomes in live yeast cells by tagging the catalytically active β1 subunit (Pre3) of the proteasome with GFP. Efficient and quantitative introduction of the β1–GFP in 20S proteasomes was confirmed by native gel electrophoresis (supplementary material Fig. S1). The GFP-labeled proteasomes had a similar distribution during starvation as reported for non-modified proteasomes previously (Laporte et al., 2008). We observed that cells in starvation show a wide heterogeneity in proteasome localization (Fig. 1A). Based on proteasome localization, we defined four localization phenotypes: (1) cells with proteasome accumulation in the nucleus (Nuclear); (2) cells displaying dots of cytoplasmic proteasome clusters (PSG); (3) cells displaying both PSGs and a nuclear accumulation of proteasomes (Nuclear + PSG); and (4) cells without any of these phenotypes, where proteasomes are approximately equally distributed between the cytoplasmic and nuclear compartments (Equal) (Fig. 1B). In a typical 5-day starvation experiment the majority of the cells are either PSG or Equal, whereas a small portion of the cells is Nuclear or Nuclear + PSG.


N-terminal acetylation and replicative age affect proteasome localization and cell fitness during aging.

van Deventer S, Menendez-Benito V, van Leeuwen F, Neefjes J - J. Cell. Sci. (2014)

Proteasome localization in nutrient-starved cells correlates with replicative age. (A) Live-cell microscopy of yeast cells in starvation shows various 20S proteasome localizations as is visualized by endogenous expression of a GFP-tagged β1 subunit (Pre3). Cells were stained with CFW to assess the replicative age of individual cells. (B) β1–GFP localization and Hoechst 33342 staining was used to define four different phenotypes: cells with cytosolic PSGs (PSG), cells with nuclear enrichment of proteasomes (Nuclear), cells that display both a nuclear enrichment of proteasomes and PSGs (Nuclear + PSG), and cells without a clear enrichment of proteasomes in PSGs or nuclei (Equal). (C) Based on CFW staining of bud scars, three different replicative age groups were defined: virgin daughter cells without bud scars (V); young mother cells with 1–2 bud scars (YM); and old mother cells with more than two bud scars (OM). (D) The prevalence of the different proteasome phenotypes in living cells from each age group was calculated by dividing the number of cells with a certain phenotype in a particular age group over the total number of cells in this age group. Results are mean±s.d. based on three independent experiments. Significance was calculated with a paired, two-tailed Student's t-test (*P<0.05, **P<0.01). Scale bars: 5 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4282048&req=5

f01: Proteasome localization in nutrient-starved cells correlates with replicative age. (A) Live-cell microscopy of yeast cells in starvation shows various 20S proteasome localizations as is visualized by endogenous expression of a GFP-tagged β1 subunit (Pre3). Cells were stained with CFW to assess the replicative age of individual cells. (B) β1–GFP localization and Hoechst 33342 staining was used to define four different phenotypes: cells with cytosolic PSGs (PSG), cells with nuclear enrichment of proteasomes (Nuclear), cells that display both a nuclear enrichment of proteasomes and PSGs (Nuclear + PSG), and cells without a clear enrichment of proteasomes in PSGs or nuclei (Equal). (C) Based on CFW staining of bud scars, three different replicative age groups were defined: virgin daughter cells without bud scars (V); young mother cells with 1–2 bud scars (YM); and old mother cells with more than two bud scars (OM). (D) The prevalence of the different proteasome phenotypes in living cells from each age group was calculated by dividing the number of cells with a certain phenotype in a particular age group over the total number of cells in this age group. Results are mean±s.d. based on three independent experiments. Significance was calculated with a paired, two-tailed Student's t-test (*P<0.05, **P<0.01). Scale bars: 5 µm.
Mentions: We visualized proteasomes in live yeast cells by tagging the catalytically active β1 subunit (Pre3) of the proteasome with GFP. Efficient and quantitative introduction of the β1–GFP in 20S proteasomes was confirmed by native gel electrophoresis (supplementary material Fig. S1). The GFP-labeled proteasomes had a similar distribution during starvation as reported for non-modified proteasomes previously (Laporte et al., 2008). We observed that cells in starvation show a wide heterogeneity in proteasome localization (Fig. 1A). Based on proteasome localization, we defined four localization phenotypes: (1) cells with proteasome accumulation in the nucleus (Nuclear); (2) cells displaying dots of cytoplasmic proteasome clusters (PSG); (3) cells displaying both PSGs and a nuclear accumulation of proteasomes (Nuclear + PSG); and (4) cells without any of these phenotypes, where proteasomes are approximately equally distributed between the cytoplasmic and nuclear compartments (Equal) (Fig. 1B). In a typical 5-day starvation experiment the majority of the cells are either PSG or Equal, whereas a small portion of the cells is Nuclear or Nuclear + PSG.

Bottom Line: By using a microscopy-based genome-wide screen, we identified genetic factors involved in these processes.Both relocalization of the proteasome and PSG formation were affected by two of the three N-acetylation complexes.These N-acetylation complexes also had different effects on the longevity of cells, indicating that each N-acetylation complex has different roles in proteasome location and aging.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute and Netherlands Proteomics Center, Plesmanlaan 121, 1066CX Amsterdam, The Netherlands.

Show MeSH
Related in: MedlinePlus