1. A Mechanism for Inhibiting the SUMO Pathway
Roberto Boggio, Riccardo Colombo, Ronald T. Hay, Giulio F. Draetta, Susanna Chiocca 
Molecular Cell 
Volume 16, Issue 4, Pages (November 2004)
DOI: /j.molcel

2. Figure 1
Gam1 Wt, but Not Gam1mt, Blocks In Vitro Sumoylation in a Dose-Dependent Manner
(A) GST RanGAP1 (substrate) was incubated with the reaction MIX (purified GST-SAE2/SAE1, GST-UBC9 in an ATP-containing buffer) with GST-SUMO-1 C52A (where indicated) and increasing amounts of HIS-Gam1 wt or HIS-Gam1mt (lanes 3 and 6, 15 ng; lanes 4 and 7, 25 ng; and lanes 5 and 8, 50 ng) for 2 hr at 30°C. Reactions were stopped as described in Experimental Procedures, loaded on SDS-PAGE, and immunoblotted.
(B) Increasing amounts of E1, but not of E2 or SUMO-1, restore the sumoylation of RanGAP1 blocked by Gam1 wt. GST RanGAP1 was incubated with the indicated amounts of purified GST-SAE2/SAE1 (+, 100 ng; ++, 200 ng; and +++, 300 ng), GST-UBC9 (+, 200 ng; ++, 400 ng; and +++, 600 ng), GST-SUMO-1 C52A (+, 2 μg; ++, 4 μg; and +++, 6 μg), and 25 ng of HIS-Gam1 wt in an ATP buffer for 2 hr at 30°C. Reactions were then stopped, loaded on SDS-PAGE, and immunoblotted.
(C) Two-step assay. In the first step, GST-SAE2/SAE1 (E1) was incubated with GST-SUMO-1 C52A in ATP buffer for 90 min at 30°C. Subsequently in the second step, GST-RanGAP-1 (substrate) was added with GST-UBC9 (E2) for 90 min at 30°C where indicated. Twenty-five nanograms of HIS-Gam1 wt (lanes 3–6) or HIS-Gam1mt (lanes 7–10) were added at the indicated addition points. One hundred and eighty minutes later, reactions were stopped, loaded on 10% SDS-PAGE, and immunoblotted. The right panel is a schematic representation of the assay.
(D) Gam1 blocks in vitro thioester bond formation between E1 and SUMO-1 in a dose-dependent manner; increasing amounts of E1, but not SUMO-1, restore the thioester bond formation blocked by Gam1.
Thioester bond formation assays were performed as described in Experimental Procedures with purified SAE2/1 (+, 0.47 μM; ++, 0.94 μM; and +++, 1.41 μM), purified SUMO-1(1–97 aa, C52A) (+, μM; ++, 26.7 μM; and +++, μM). HIS-Gam1 wt was added to the reaction where indicated (lane 7, μM; lane 8, μM; lanes 9 and 11–15, 0.78 μM). Reaction products were stopped as described, loaded on SDS-PAGE, and immunoblotted.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 Gam1 Is Not a Sumo Protease
(A) Isopeptidase activity: GST-RanGAP1 recombinant purified protein (2 μg) was sumoylated in vitro, and 2 μg of the yeast SUMO protease GST-Ulp1 (lane 2) or GST-Gam1 (lane 3) were then added to the sumoylated protein for 1 hr at 30°C.
(B) C-terminal hydrolase activity: GST-SUMO-1-FLAG (3 μg) was used as a substrate plus either GST-Ulp1 (lane 2) or GST-Gam1(lane 3) (2 μg) for 1 hr at 30°C.
(C and D) HeLa cells were transfected with the indicated plasmids. Forty-eight hours after transfection, 30 μg of SDS lysates were processed for Western blot analysis.
(E) HeLa cells were transfected with the indicated plasmids. Forty-eight hours after transfection, cells were lysed in E1A buffer and processed as described in the right panel (2 μg GST-SP3 were used). The right panel is a schematic representation of the assay.
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Gam1 Leads to the Disappearance of Endogenous SAE1, SAE2, and UBC9
(A) HeLa cells were transfected by using plasmids directing the expression of GFP together with the indicated plasmids. Twenty-four hours after transfection, GFP-positive cells were sorted and then processed for western blot analysis.
(B) HeLa cells were transfected with the indicated plasmids, and expression levels of the indicated mRNAs were determined by real-time PCR as described in Experimental Procedures. Protein expression was monitored by Western blot analysis.
(C) LMH cells were infected with 300 particles per cell of CELO virus. After 2 days at 37°C, 30 μg of lysate protein was analyzed by immunoblotting.
(D) Pulse-chase analysis of SAE1 and UBC9 turnover rate. Phoenix cells were transfected with the indicated plasmids, and 24 hr later, cells were labeled with [35S] methionine for 40 min and chased with cold medium for the indicated times. Extracts were then subjected to immunoprecipitation with the indicated antibodies and analyzed by immunoblotting. Because cells were not sorted, we could not observe complete SAE1 and UBC9 reduction. Input, 30 μg.
(E) HeLa cells were transfected by using the plasmids directing the expression of GFP together with the indicated plasmids. Twenty-four hours after transfection GFP-positive cells were sorted and then processed for Western blot analysis. MG132 (final concentration of 10 μM) or DMSO was added at the beginning of the transfection. Thirty micrograms of total extracts of indicated transfected and sorted cells were loaded on SDS-PAGE and immunoblotted.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
Gam1 Binds E1 (SAE1/SAE2) Enzyme In Vivo and In Vitro and Is Found in the Same Complex as E1
(A and B) In vitro reactions were carried out as described in Experimental Procedures. The samples were loaded in 10% SDS-PAGE and immunoblotted with the indicated antibodies. Five hundred nanograms of purified SAE1 or SAE2 were loaded as input.
(C) HeLa cells were transfected with the indicated plasmids and lysed in E1A buffer 2 days later. 1.5 mg of total extracts of indicated transfected cells were immunoprecipitated (IP) in E1A buffer with the indicated antibodies, electrophoresed on SDS-PAGE, and immunoblotted (WB) with the indicated antibodies. Thirty micrograms of total extracts of indicated transfected cells were loaded as input.
(D) HeLa cells were transfected with the indicated plasmids and lysed in RIPA buffer 2 days later, and a sequential immunoprecipitation was carried out from 20 mg of crude extract as depicted in the right panel. Input, 30 μg.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 Gam1 Wt Inactivates E1 In Vivo
(A) HeLa cells were transfected with the indicated plasmids, lysed in E1A buffer 2 days later, and processed as depicted in the right panel with 50 ng of E2, 2 μg of SUMO, and 330 ng of substrate.
(B) HeLa cells were transfected with the indicated plasmids and lysed in E1A buffer 2 days later. Eight hundred micrograms of total extracts of indicated transfected cells were immunoprecipitated (IP) in RIPA buffer with the indicated antibodies, electrophoresed on SDS-PAGE, and immunoblotted (WB) with the indicated antibodies.
(C) HeLa cells were transfected with the indicated plasmids, lysed in E1A buffer 2 days later, and processed as depicted in the right panel with 50 ng of E2, 2 μg of SUMO, and 330 ng of substrate.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6 Gam1, Transcription, and Sumoylation
(A) HeLa cells were transfected with 0.5 μg of RSV-Luc reporter plasmid together with 1 μg of the indicated plasmids. After 24 hr, 30 μg of E1A-buffer cellular lysate was used for luciferase assay. Data, expressed in luciferase units, are the average of three independent experiments with error bars representing the standard deviation. The lower panel shows an example of the expression levels of the transfected proteins.
(B) HeLa cells were transfected with 0.3 μg of RSV-Luc reporter plasmid together with 0.5 μg of the indicated plasmids (each transfection was normalized as described in Experimental Procedures). After 24 hr, 30 μg of E1A-buffer cellular lysate was used for luciferase assay. Results are expressed as fold induction of luciferase units in transfected cells where Gam1 is present versus luciferase units in cells transfected with the same vectors but without Gam1 and are the average of three independent experiments with error bars representing the standard deviation.
(C) Gam1 activates SP3 transcriptional activity. HeLa cells were transfected with 0.5 μg of 5Gal4 E1B-Luc reporter plasmid together with 1 μg of the indicated plasmids (each transfection was normalized as described in Experimental Procedures). After 48 hr, 30 μg of E1A-buffer cellular lysate was used for luciferase assay. Results are expressed as fold induction of luciferase units in transfected cells where Gam1 is present versus luciferase units in cells transfected with the same vectors but without Gam1 and are the average of three independent experiments with error bars representing the standard deviation.
(D) Sumoylation of SP3: SAE1 and SAE2 reverse the effect of Gam1. HeLa cells were transfected with the indicated plasmids and lysed in SDS buffer 2 days later. Thirty micrograms of total extracts of indicated transfected cells were analyzed by immunoblotting.
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7 A Schematic Model for the Role of Gam1
The sumoylation pathway in the absence (left) or presence of Gam1 (right). Gam1 binds SAE1/SAE2, induces SAE1/SAE2 and UBC9 degradation, blocks the sumoylation pathway, and activates transcription.
Molecular Cell  , DOI: ( /j.molcel )