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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, Universität 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.


Related in: MedlinePlus

Localization of rice SAUL-type PUB-ARM proteins at the plasma membrane. (A) Localization of GFP-OsPUB23 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB23 proteins depicted in green at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (B) Localization of AtINT4-RFP fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of AtINT4-RFP proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (C) Merged image of (A,B). Yellow signals derived from the overlap of green and red fluorescences from (A,B). (D) Bright filed image of the protoplast analyzed in (A–C). (E) Localization of GFP-OsPUB21.1 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green at the plasma membrane of transformed protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (F) Localization of GFP-OsPUB21.2 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green in the cytosol of transformed Arabidopsis protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red.
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Figure 2: Localization of rice SAUL-type PUB-ARM proteins at the plasma membrane. (A) Localization of GFP-OsPUB23 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB23 proteins depicted in green at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (B) Localization of AtINT4-RFP fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of AtINT4-RFP proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (C) Merged image of (A,B). Yellow signals derived from the overlap of green and red fluorescences from (A,B). (D) Bright filed image of the protoplast analyzed in (A–C). (E) Localization of GFP-OsPUB21.1 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green at the plasma membrane of transformed protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (F) Localization of GFP-OsPUB21.2 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green in the cytosol of transformed Arabidopsis protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red.

Mentions: Plasma membrane-association was observed for the two SAUL1 paralogs AtPUB42 and AtPUB43, but not for any other member of the Arabidopsis PUB-ARM protein family (Drechsel et al., 2011). For SAUL1 it has been demonstrated that this specific localization depends on the additional ARM repeat domain in the C-terminus that is unique to SAUL1, AtPUB42, and AtPUB43. The domain structure of AtPUB43 is schematically depicted in Figure 1A. To test whether the C-terminal ARM repeat domain is also essential for plasma membrane-association of AtPUB43, we analyzed the localization of fusion proteins between GFP and truncated AtPUB43 proteins by confocal laser scanning microscopy on transformed Arabidopsis protoplasts. As for SAUL1, deletion of the N-terminal part of AtPUB43 in AtPUB43ΔARM1−6-GFP proteins did not result in the loss of plasma membrane-association (Figure 1B). This deletion protein, however, was not equally distributed in the plasma membrane, but occurred in large patches (Figures 1B,C). A similar pattern has been observed for SAUL1 proteins with truncated and/or masked N-terminus previously (Drechsel et al., 2011). In a next step, either ARM repeats 7–12 or 10–12 were deleted in AtPUB43. Both proteins, AtPUB43ΔARM7−12-GFP and AtPUB43ΔARM10−12-GFP, were localized to the cytoplasm and not to the plasma membrane (Figures 1D,E). These data indicated that indeed the C-terminal ARM repeat domain is essential and sufficient for plasma membrane-association of AtPUB43 and that this is a general feature of SAUL1-type E3 ubiquitin ligases. The analysis of transgenic Arabidopsis plants overexpressing GFP-AtPUB43ΔARM1−6 showed that the C-terminus by itself had no effect on plant growth and development (Figure S1). To test whether masking the N-terminus of SAUL1-type proteins, which eventually leads to patchy distribution of the protein at the plasma membrane (Drechsel et al., 2011 and Figure 2E), may affect growth and development, we analyzed CaMV35S::YFP-SAUL1 plants. Indeed, overexpression of YFP-SAUL1 fusion proteins resulted in a severe growth defect when compared to growth of wildtype plants (Figures 1F,G).


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 rice SAUL-type PUB-ARM proteins at the plasma membrane. (A) Localization of GFP-OsPUB23 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB23 proteins depicted in green at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (B) Localization of AtINT4-RFP fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of AtINT4-RFP proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (C) Merged image of (A,B). Yellow signals derived from the overlap of green and red fluorescences from (A,B). (D) Bright filed image of the protoplast analyzed in (A–C). (E) Localization of GFP-OsPUB21.1 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green at the plasma membrane of transformed protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (F) Localization of GFP-OsPUB21.2 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green in the cytosol of transformed Arabidopsis protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red.
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Figure 2: Localization of rice SAUL-type PUB-ARM proteins at the plasma membrane. (A) Localization of GFP-OsPUB23 fusion proteins at the plasma membrane of Arabidopsis protoplasts. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB23 proteins depicted in green at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (B) Localization of AtINT4-RFP fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of AtINT4-RFP proteins depicted in red at the plasma membrane of protoplasts co-expressing GFP-OsPUB23 and AtINT4-RFP. Chlorophyll auto-fluorescence within the chloroplasts is depicted in blue. (C) Merged image of (A,B). Yellow signals derived from the overlap of green and red fluorescences from (A,B). (D) Bright filed image of the protoplast analyzed in (A–C). (E) Localization of GFP-OsPUB21.1 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green at the plasma membrane of transformed protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red. (F) Localization of GFP-OsPUB21.2 fusion proteins at the plasma membrane. Confocal laser scanning microscopy detected fluorescence of GFP-OsPUB21.1 proteins depicted in green in the cytosol of transformed Arabidopsis protoplasts. Chlorophyll auto-fluorescence within the chloroplasts is depicted in red.
Mentions: Plasma membrane-association was observed for the two SAUL1 paralogs AtPUB42 and AtPUB43, but not for any other member of the Arabidopsis PUB-ARM protein family (Drechsel et al., 2011). For SAUL1 it has been demonstrated that this specific localization depends on the additional ARM repeat domain in the C-terminus that is unique to SAUL1, AtPUB42, and AtPUB43. The domain structure of AtPUB43 is schematically depicted in Figure 1A. To test whether the C-terminal ARM repeat domain is also essential for plasma membrane-association of AtPUB43, we analyzed the localization of fusion proteins between GFP and truncated AtPUB43 proteins by confocal laser scanning microscopy on transformed Arabidopsis protoplasts. As for SAUL1, deletion of the N-terminal part of AtPUB43 in AtPUB43ΔARM1−6-GFP proteins did not result in the loss of plasma membrane-association (Figure 1B). This deletion protein, however, was not equally distributed in the plasma membrane, but occurred in large patches (Figures 1B,C). A similar pattern has been observed for SAUL1 proteins with truncated and/or masked N-terminus previously (Drechsel et al., 2011). In a next step, either ARM repeats 7–12 or 10–12 were deleted in AtPUB43. Both proteins, AtPUB43ΔARM7−12-GFP and AtPUB43ΔARM10−12-GFP, were localized to the cytoplasm and not to the plasma membrane (Figures 1D,E). These data indicated that indeed the C-terminal ARM repeat domain is essential and sufficient for plasma membrane-association of AtPUB43 and that this is a general feature of SAUL1-type E3 ubiquitin ligases. The analysis of transgenic Arabidopsis plants overexpressing GFP-AtPUB43ΔARM1−6 showed that the C-terminus by itself had no effect on plant growth and development (Figure S1). To test whether masking the N-terminus of SAUL1-type proteins, which eventually leads to patchy distribution of the protein at the plasma membrane (Drechsel et al., 2011 and Figure 2E), may affect growth and development, we analyzed CaMV35S::YFP-SAUL1 plants. Indeed, overexpression of YFP-SAUL1 fusion proteins resulted in a severe growth defect when compared to growth of wildtype plants (Figures 1F,G).

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, Universität 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.


Related in: MedlinePlus