Limits...
Plasma membrane-association of SAUL1-type plant U-box armadillo repeat proteins is conserved in land plants.

Vogelmann K, Subert C, Danzberger N, Drechsel G, Bergler J, Kotur T, Burmester T, Hoth S - Front Plant Sci (2014)

Bottom Line: Phylogenetic analysis identified orthologs of SAUL1 in these plant species.Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects.Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, Biozentrum Klein Flottbek, Universit├Ąt Hamburg Hamburg, Germany.

ABSTRACT
Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box (PUB) armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

No MeSH data available.


Localization of SAUL-type PUB-ARM proteins from Populus trichocarpa at the plasma membrane. (A) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0005s27480 fusion proteins. GFP fluorescence depicted in green was detected at the plasma membrane in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (B) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0004s02840 fusion proteins. GFP fluorescence depicted in green was detected in cytosol and nucleus in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (C) Localization of GFP-Pt0005s27480 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-Pt0005s27480 proteins depicted in green (left) and RFP-UmSrt1 (middle) proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-Pt0005s27480 and RFP-UmSrt1. Merging both images resulted in yellow signals (right) derived from the overlap of green (GFP-Pt0005s27480) and red (RFP-UmSrt1) fluorescences. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3928556&req=5

Figure 4: Localization of SAUL-type PUB-ARM proteins from Populus trichocarpa at the plasma membrane. (A) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0005s27480 fusion proteins. GFP fluorescence depicted in green was detected at the plasma membrane in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (B) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0004s02840 fusion proteins. GFP fluorescence depicted in green was detected in cytosol and nucleus in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (C) Localization of GFP-Pt0005s27480 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-Pt0005s27480 proteins depicted in green (left) and RFP-UmSrt1 (middle) proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-Pt0005s27480 and RFP-UmSrt1. Merging both images resulted in yellow signals (right) derived from the overlap of green (GFP-Pt0005s27480) and red (RFP-UmSrt1) fluorescences. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue.

Mentions: Two PUB-ARM proteins from each organism, one SAUL1-type and one without the additional C-terminal ARM repeat domain, were selected to investigate conservation of SAUL1-type plasma membrane-association in Populus trichocarpa and Physcomitrella patens. Confocal laser scanning microscopy on transformed Arabidopsis leaf cell protoplasts and tobacco leaf epidermal cells analyzed localization of GFP fusion proteins. The poplar SAUL1-type PUB-ARM protein Pt0005s27480 (POPTR_0005s27480) localized to the plasma membrane as indicated by GFP fluorescence of GFP-Pt0005s27480 (Figure 4A) or Pt0005s27480-GFP (not shown) fusion proteins. In contrast, the same approach showed that the poplar PUB-ARM protein Pt0004s02840 (POPTR_0004s02840) that is lacking the C-terminal ARM repeat domain was localized to cytoplasm and nucleus (Figure 4B and not shown). The same results were obtained in Physcomitrella patens. Whereas fusion proteins with the SAUL1-type PUB-ARM protein Pp1s3_414V6 were associated with the plasma membrane, the Pp1s67_203V6 protein that also does not contain the additional ARM repeat domain localized to cytoplasm and nucleus (Figures 5A,B). To confirm localization of Pt0005s27480 and Pp1s3_414V6 at the plasma membrane, both GFP fusion proteins were co-expressed with the plasma membrane sugar transport protein UmSrt1 from the phytopathogenic fungus Ustilago maydis fused to RFP (Figures 4C, 5C). The UmSrt1 transport protein was localized to the plasma membrane as described previously (c.f. Drechsel et al., 2011). Merging green and red fluorescence of GFP-Pt0005s27480 and RFP-UmSrt1 (Figure 4C) or GFP-Pp1s3_414V6 and RFP-UmSrt1 (Figure 5C) resulted in yellow signals and clearly showed that both SAUL1-type proteins are associated to the plasma membrane.


Plasma membrane-association of SAUL1-type plant U-box armadillo repeat proteins is conserved in land plants.

Vogelmann K, Subert C, Danzberger N, Drechsel G, Bergler J, Kotur T, Burmester T, Hoth S - Front Plant Sci (2014)

Localization of SAUL-type PUB-ARM proteins from Populus trichocarpa at the plasma membrane. (A) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0005s27480 fusion proteins. GFP fluorescence depicted in green was detected at the plasma membrane in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (B) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0004s02840 fusion proteins. GFP fluorescence depicted in green was detected in cytosol and nucleus in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (C) Localization of GFP-Pt0005s27480 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-Pt0005s27480 proteins depicted in green (left) and RFP-UmSrt1 (middle) proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-Pt0005s27480 and RFP-UmSrt1. Merging both images resulted in yellow signals (right) derived from the overlap of green (GFP-Pt0005s27480) and red (RFP-UmSrt1) fluorescences. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3928556&req=5

Figure 4: Localization of SAUL-type PUB-ARM proteins from Populus trichocarpa at the plasma membrane. (A) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0005s27480 fusion proteins. GFP fluorescence depicted in green was detected at the plasma membrane in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (B) Confocal analysis of fluorescence of Arabidopsis leaf protoplasts (top panel) and tobacco leaf epidermal cells (bottom panel) expressing GFP-Pt0004s02840 fusion proteins. GFP fluorescence depicted in green was detected in cytosol and nucleus in both cell types. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (C) Localization of GFP-Pt0005s27480 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-Pt0005s27480 proteins depicted in green (left) and RFP-UmSrt1 (middle) proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-Pt0005s27480 and RFP-UmSrt1. Merging both images resulted in yellow signals (right) derived from the overlap of green (GFP-Pt0005s27480) and red (RFP-UmSrt1) fluorescences. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue.
Mentions: Two PUB-ARM proteins from each organism, one SAUL1-type and one without the additional C-terminal ARM repeat domain, were selected to investigate conservation of SAUL1-type plasma membrane-association in Populus trichocarpa and Physcomitrella patens. Confocal laser scanning microscopy on transformed Arabidopsis leaf cell protoplasts and tobacco leaf epidermal cells analyzed localization of GFP fusion proteins. The poplar SAUL1-type PUB-ARM protein Pt0005s27480 (POPTR_0005s27480) localized to the plasma membrane as indicated by GFP fluorescence of GFP-Pt0005s27480 (Figure 4A) or Pt0005s27480-GFP (not shown) fusion proteins. In contrast, the same approach showed that the poplar PUB-ARM protein Pt0004s02840 (POPTR_0004s02840) that is lacking the C-terminal ARM repeat domain was localized to cytoplasm and nucleus (Figure 4B and not shown). The same results were obtained in Physcomitrella patens. Whereas fusion proteins with the SAUL1-type PUB-ARM protein Pp1s3_414V6 were associated with the plasma membrane, the Pp1s67_203V6 protein that also does not contain the additional ARM repeat domain localized to cytoplasm and nucleus (Figures 5A,B). To confirm localization of Pt0005s27480 and Pp1s3_414V6 at the plasma membrane, both GFP fusion proteins were co-expressed with the plasma membrane sugar transport protein UmSrt1 from the phytopathogenic fungus Ustilago maydis fused to RFP (Figures 4C, 5C). The UmSrt1 transport protein was localized to the plasma membrane as described previously (c.f. Drechsel et al., 2011). Merging green and red fluorescence of GFP-Pt0005s27480 and RFP-UmSrt1 (Figure 4C) or GFP-Pp1s3_414V6 and RFP-UmSrt1 (Figure 5C) resulted in yellow signals and clearly showed that both SAUL1-type proteins are associated to the plasma membrane.

Bottom Line: Phylogenetic analysis identified orthologs of SAUL1 in these plant species.Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects.Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

View Article: PubMed Central - PubMed

Affiliation: Molekulare Pflanzenphysiologie, Biozentrum Klein Flottbek, Universit├Ąt Hamburg Hamburg, Germany.

ABSTRACT
Post-translational protein modification plays a pivotal role in the regulation and specific turnover of proteins. One of these important modifications is the ubiquitination of target proteins, which can occur at distinct cellular compartments. At the plasma membrane, ubiquitination regulates the internalization and thus trafficking of membrane proteins such as receptors and channels. The Arabidopsis plant U-box (PUB) armadillo repeat (PUB-ARM) ubiquitin ligase SAUL1 (SENESCENCE-ASSOCIATED UBIQUITIN LIGASE1) is part of the ubiquitination machinery at the plasma membrane. In contrast to most other PUB-ARM proteins, SAUL1 carries additional C-terminal ARM repeats responsible for plasma membrane-association. Here, we demonstrated that the C-terminal ARM repeat domain is also essential and sufficient to mediate plasma membrane-association of the closest Arabidopis paralog AtPUB43. We investigated targeting of PUB-ARM ubiquitin ligases of different plant species to find out whether plasma membrane-association of SAUL1-type PUB-ARM proteins is conserved. Phylogenetic analysis identified orthologs of SAUL1 in these plant species. Intracellular localization of transiently expressed GFP fusion proteins revealed that indeed plasma membrane-association due to additional C-terminal ARM repeats represents a conserved feature of SAUL1-type proteins. Analyses of transgenic Arabidopsis plants overexpressing N-terminally masked or truncated proteins revealed that interfering with the function of SAUL1-type proteins resulted in severe growth defects. Our results suggest an ancient origin of ubiquitination at the plasma membrane in the evolution of land plants.

No MeSH data available.