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Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

Nie M, Xie Y, Loo JA, Courey AJ - PLoS ONE (2009)

Bottom Line: For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation.We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody.Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

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A fly SUMO-ome: characterization and validation.A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His)6-FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The QSUMO vector, which encodes the mature form of SUMO (SUMOGG) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, QΔGG, which expresses a conjugation defective form of SUMO (SUMOΔGG), was used in place of QSUMO. C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with QSUMO or QΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His)6- tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).
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pone-0005905-g001: A fly SUMO-ome: characterization and validation.A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His)6-FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The QSUMO vector, which encodes the mature form of SUMO (SUMOGG) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, QΔGG, which expresses a conjugation defective form of SUMO (SUMOΔGG), was used in place of QSUMO. C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with QSUMO or QΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His)6- tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).

Mentions: To determine the early embryonic SUMO-ome (catalog of sumoylated proteins), we adopted a scheme that involved a two-step affinity purification strategy using SUMO tagged at its N-terminus with both (His)6 and FLAG tags, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein identification of trypsin-digested proteins (Figure 1A). We initially attempted to express tagged SUMO using modified sumo genomic clones, but were unsuccessful presumably due to the need for unknown distant cis-regulatory modules to direct sumo expression. We therefore turned to the Gal4-UAS system [36], and drove ubiquitous maternal expression of tagged SUMO at levels slightly lower than that of endogenous SUMO (Figure S1). Tagged SUMO rescues the lethality resulting from sumo mutations (data not shown) demonstrating the functionality of the tagged protein.


Genetic and proteomic evidence for roles of Drosophila SUMO in cell cycle control, Ras signaling, and early pattern formation.

Nie M, Xie Y, Loo JA, Courey AJ - PLoS ONE (2009)

A fly SUMO-ome: characterization and validation.A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His)6-FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The QSUMO vector, which encodes the mature form of SUMO (SUMOGG) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, QΔGG, which expresses a conjugation defective form of SUMO (SUMOΔGG), was used in place of QSUMO. C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with QSUMO or QΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His)6- tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).
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Related In: Results  -  Collection

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pone-0005905-g001: A fly SUMO-ome: characterization and validation.A) Scheme for identifying Drosophila embryonic SUMO conjugates. SUMO conjugates were isolated by tandem affinity purification from transgenic fly embryos expressing (His)6-FLAG-SUMO. The initial purification step (Ni-NTA chromatography) was performed under denaturing conditions. To maximize the chance of detecting low abundance proteins in the complex protein mixture, the affinity-purified proteins were separated by SDS-PAGE, and the lane was cut into 20 evenly divided gel slices. Tryptic peptides extracted from each gel slice were analyzed by LC-MS/MS. B) A bacterial sumoylation assay. The QSUMO vector, which encodes the mature form of SUMO (SUMOGG) along with SAE1, SAE2, and Ubc9 expressed from separate T7/lac promoters, was used in combination with a vector expressing a GST-tagged candidate substrate. As a negative control, QΔGG, which expresses a conjugation defective form of SUMO (SUMOΔGG), was used in place of QSUMO. C) Bacterial sumoylation assays were used to validate proteins identified in the proteomic screen as sumoylation substrates. GST-tagged candidate SUMO conjugates were expressed in BL21 cells co-transformed with QSUMO or QΔGG vectors, purified using glutathione beads, and immunoblotted using antibodies against GST, SUMO, or poly-His (to detect 6xHis-tagged SUMO). GST by itself was not sumoylated in this assay. Black arrows point to the bands representing sumoylated proteins, and open arrow points to a non-specific reacting band. D) The eIF4E protein was purified from Drosophila S2 cells stably expressing FLAG-(His)6- tagged eIF4E using Ni-NTA beads under denaturing conditions. The resulting proteins were probed with anti-FLAG antibody in a Western blot. The cells were treated with SUMO or control YFP dsRNA for 3 days prior to cell lysis. In the control sample, the bands representing the sumoylated species (black arrows) have intensities that are 8.1% (top) and 12.9% (bottom) of the intensity of the band representing unmodified eIF4E (∼40 kDa), whereas in the SUMO knockdown sample, they are reduced to 1.8% (top) and 3.5% (bottom). Quantitation was performed using Quantity One 4.3.0 (BioRad).
Mentions: To determine the early embryonic SUMO-ome (catalog of sumoylated proteins), we adopted a scheme that involved a two-step affinity purification strategy using SUMO tagged at its N-terminus with both (His)6 and FLAG tags, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protein identification of trypsin-digested proteins (Figure 1A). We initially attempted to express tagged SUMO using modified sumo genomic clones, but were unsuccessful presumably due to the need for unknown distant cis-regulatory modules to direct sumo expression. We therefore turned to the Gal4-UAS system [36], and drove ubiquitous maternal expression of tagged SUMO at levels slightly lower than that of endogenous SUMO (Figure S1). Tagged SUMO rescues the lethality resulting from sumo mutations (data not shown) demonstrating the functionality of the tagged protein.

Bottom Line: For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation.We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody.Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, California, United States of America.

ABSTRACT
SUMO is a protein modifier that is vital for multicellular development. Here we present the first system-wide analysis, combining multiple approaches, to correlate the sumoylated proteome (SUMO-ome) in a multicellular organism with the developmental roles of SUMO. Using mass-spectrometry-based protein identification, we found over 140 largely novel SUMO conjugates in the early Drosophila embryo. Enriched functional groups include proteins involved in Ras signaling, cell cycle, and pattern formation. In support of the functional significance of these findings, sumo germline clone embryos exhibited phenotypes indicative of defects in these same three processes. Our cell culture and immunolocalization studies further substantiate roles for SUMO in Ras signaling and cell cycle regulation. For example, we found that SUMO is required for efficient Ras-mediated MAP kinase activation upstream or at the level of Ras activation. We further found that SUMO is dynamically localized during mitosis to the condensed chromosomes, and later also to the midbody. Polo kinase, a SUMO substrate found in our screen, partially colocalizes with SUMO at both sites. These studies show that SUMO coordinates multiple regulatory processes during oogenesis and early embryogenesis. In addition, our database of sumoylated proteins provides a valuable resource for those studying the roles of SUMO in development.

Show MeSH
Related in: MedlinePlus