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Expression and regulation of the mammalian SUMO-1 E1 enzyme¹

Laboratory of Molecular Embryology, NICHD, National Institutes of Health, Bethesda, Maryland 20892-5431, USA

³Correspondence: Laboratory of Molecular Embryology, NICHD, NIH, Bldg. 18, Room 106, Bethesda, MD 20892-5431, USA. Presently in the Laboratory of Gene Regulation and Development (same address). E-mail: mdasso{at}helix.nih.gov

SPECIFIC AIMS

SUMO-1 E1 enzyme subunits associate as a simple heterodimeric complex, but purified E1 heterodimer plus the E2 enzyme is significantly less efficient than cellular extracts in catalyzing SUMO-1 conjugation, suggesting the existence of previously uncharacterized positive regulators of this reaction. In conjunction with further examination of the expression, localization, and biochemical behavior of SUMO-1 pathway enzymes, these findings suggest that SUMO-1 conjugation may be controlled during the cell cycle by at least two separate mechanisms.

PRINCIPAL FINDINGS

1. SUMO-1 conjugation varies during the cell cycle
Analysis of three prominent SUMO-1-conjugated species in HeLa cells revealed distinct patterns of abundance in cells synchronized in different parts of the cell cycle (Fig. 1A ). The 90 kDa species corresponding to RanGAP1-SUMO-1 was roughly constant throughout the cell cycle. The abundance of the conjugated species with an apparent molecular mass of 100 kDa (p100) changed less than twofold, being most abundant in S phase and less abundant in G₁ phase. The conjugated species with an apparent molecular mass of 160 kDa (p160) showed a significant change in abundance, peaking with high levels during mid-S phase. These data show that the abundance of some SUMO-1-conjugated species varies specifically during the cell cycle, suggesting that the metazoan SUMO-1 conjugation pathway is subject to cell cycle regulation.

View larger version (40K): [in this window] [in a new window]   Figure 1. Cell Cycle analysis and subcellular localization of hAos1, hUba2 proteins. A) SUMO-1-conjugated species during the cell cycle. The left panel shows a Western blot of asynchronous HeLa cells. Molecular markers are indicated on the left and the primary conjugation products are indicated on the right. HeLa cells were synchronized using an aphidicolin and nocodazole block-release protocol. Western blot analysis with anti-SUMO-1 antibodies was performed on samples harvested at the indicated times. The relative amounts of the major conjugated species were quantitated at each point by densitometric analysis. B) Abundance of hUba2 and hAos1 proteins in the cell cycle. HeLa cells were synchronized with aphidicolin at the G1/S transition. At each time point, cells were harvested and analyzed in triplicate by sequential Western blotting with anti-mAos1, anti-mUba2, and anti-Ran (Transduction Laboratories, Lexington, KY). The strength of signals was measured by PhosphorImager analysis and normalized with respect to the signal from Ran. C) HeLa cells were stained with antibodies directed against mUba2 and mAos1 as indicated and visualized with FITC-conjugated secondary antibodies (left). For each experiment, DNA staining with Hoechst 33342 dye (middle) and phase images (right) are shown.

2. hAos2 expression is regulated in a tissue-specific, cell cycle-dependent manner
We cloned murine homologues of Aos1 and Uba2, raising antibodies against each of these proteins. In this report, the mouse homologues of the Aos1 and Uba2 proteins will be designated as mAos1 and mUba2, respectively. Similarly, the human proteins will be designated as hAos1 and hUba2. Western blotting analysis of synchronized HeLa cells using anti-Uba2, -Aos1, and -Ubc9 antibodies did not show substantial changes in the concentrations of hUba2 (Fig. 1B ) or the SUMO-1-conjugating enzyme hUbc9 (data not shown) proteins during the cell cycle. In contrast, the level of hAos1 protein increased as cells progressed through S phase, followed by a substantial decrease in G₂ phase. Since the increased abundance of the p160 species correlates with increased hAos1 levels, these data suggest that regulation of hAos1 protein concentrations during S phase might provide a mechanism whereby SUMO-1 conjugation could be controlled. We also examined the expression of Aos1, Uba2, and Ubc9 mRNAs in different mouse tissues by Northern blot analysis. Detectable levels of each mRNA were present in brain, thymus, heart, lung, testis, and ovary as well as in embryonic tissue (see full text online), indicating that the SUMO-1 pathway is functional in most organs, although the relative level of expression for different pathway components varies among tissues.

3. hAos1 and hUba2 localize to the nucleus
We performed indirect immunofluorescence in HeLa cells using affinity-purified anti-Aos1 and anti-Uba2 antibodies (Fig. 1C ). We found that hAos1 and hUba2 proteins are distributed throughout nuclei but excluded from nucleoli. It has been shown elsewhere that hUbc9 distributes throughout the nucleus and at nuclear pores. These results are consistent with the idea that most SUMO-1 conjugation occurs within nuclei.

4. The predominant form of hUba2 and hAos1 is a simple heterodimer
Yeast Uba2p associates with multiple proteins in addition to Aos1p. We therefore examined the biochemical properties of hUba2 and hAos1 during subcellular fractionation of HeLa cells and column chromatography tissue (see full text online). Both proteins were fully extracted from lysed HeLa cells with low salt buffers and neither protein was tightly attached to insoluble cellular structures, such as the nuclear matrix. Some hUbc9 remains in the postextraction pellet, possibly in association with the RanBP2 protein in nuclear pores. When low-salt extracts from asynchronous HeLa cells were further fractionated by gel filtration chromatography, hAos1 and hUba2 comigrated in fractions corresponding to 150–160 kDa protein complexes, suggesting that the majority of hUba2 and hAos1 in asynchronous cells form a simple heterodimer. The bulk of hUba2 and hAos1 also closely comigrated in a series of other column chromatography procedures, including Mono Q and SUMO-1 affinity columns, further supporting the notion that each protein is tightly complexed with the other. These data argue against the association of a large fraction hAos1 or hUba2 with other abundant complexes.

5. Additional factors are required for efficient SUMO-1 conjugation
It has been reported that the hAos1/hUba2 complex in combination with hUbc9 is sufficient for conjugation in vitro. To evaluate whether there may be additional cellular cofactors that regulate SUMO-1 conjugation, we purified hAos1/hUba2 to homogeneity (Fig. 2A ). This fraction was active as an activating (E1) enzyme for SUMO-1 in a reaction containing ATP, recombinant hUbc9p, recombinant SUMO-1, and a model RanGAP1 substrate (RanGAP-C2) (Fig. 2B ). Although in vitro reactions reconstituted in this manner were able to conjugate RanGAP-C2, they were significantly less efficient than reactions containing either HeLa cell extracts (Fig. 2C ) or Xenopus egg extracts (data not shown). Notably, in vitro reactions required 100-fold more recombinant hUbc9 protein than HeLa cell extracts to achieve the same level of conjugation.

View larger version (21K): [in this window] [in a new window]   Figure 2. Analysis of SUMO-1 conjugation in vitro. A) The hUba2/hAos1 complex was purified by chromatography over Mono Q, gel filtration, and SUMO-1 affinity resins. The final fraction was separated on SDS-PAGE and visualized by silver staining. Positions of hUba2 and hAos1 are indicated on the right, molecular mass markers are indicated on the left. B) The conjugation of RanGAP1-C2 was performed in reactions containing or omitting ATP, hUbc9p, and wild-type SUMO-1 (SUMO1G97), as indicated. As controls, inactive forms of hUbc9 (Ubc9-C93R,L97A) and SUMO-1 (SUMO1G96, lacking a carboxyl-terminal di-glycine motif) were included in similar reactions, but these inactive forms did not support RanGAP1-C2 conjugation. RanGAP1-C2 conjugation was monitored using an antibody directed against the T7-tag of RanGAP-C2 as the accumulation of forms of the RanGAP-C2 with a reduced electrophoretic mobility. C) The SUMO-1 conjugation reaction was performed in the presence of the indicated concentrations of hUbc9 (lower panel). The concentration of hUbc9 in the reaction mixture was confirmed by anti-Ubc9 immunoblotting (upper panel). SUMO-1 conjugation with purified proteins required 100-fold more hUbc9 protein than that found in crude extract to achieve similar conjugation efficiencies. D) Diluted HeLa cell extract increases conjugation activity of the reconstituted reaction. The conjugation reaction was performed with the volume of crude cell extract indicated in either the presence or absence of both purified E1 and hUbc9.

It is possible that this lower level of conjugation reflects a lower activity of recombinant hUbc9 protein. However, we do not favor this explanation, since partially purified fractions of hUbc9 from HeLa cells behaved equivalently to the bacterially expressed hUbc9 protein and were unable to reconstitute conjugation at the level observed in crude extracts. Moreover, a small amount of HeLa extract augmented conjugation by the purified proteins in a manner disproportionate to its Ubc9 content (Fig. 2D ). Together, these findings suggest that there may be an additional activator(s) in crude cellular extracts that promotes SUMO-1 conjugation.

CONCLUSIONS

Many questions remain to be addressed regarding how SUMO-1 conjugation is regulated. We found that the abundance of SUMO-1-conjugated species varies during the cell cycle. We investigated the expression, subcellular localization, and interactions of hAos1 and hUba2, as well as their function in in vitro assays. Our data suggest two likely points at which this pathway could be controlled: 1) the abundance of E1 enzyme subunits, particularly the hAos1 protein, and 2) the activity of accessory factors, which enhances the conjugation reaction in vitro.

One obvious difference between the ubiquitin and SUMO-1 pathways is that the E1 enzyme for SUMO-1 is a heterodimer whereas the ubiquitin E1 enzyme is a single polypeptide. The heterodimeric structure of the SUMO-1 E1 enzyme lends itself to several modes of regulation, particularly by modulating the localization or abundance of the subunits separately or by controlling their association to each other. hUba2 and hAos1 show similar patterns of localization, as judged by immunofluorescent staining (Fig. 1C ). Thus, we saw no evidence that their localization was separately regulated. This nuclear localization was also notable because some SUMO-1 targets are cytosolic. Our findings would imply either that conjugation of these targets uses a very small cytosolic pool of hUba2/hAos1 or that conjugation involves trafficking of SUMO-1 enzymes or substrates across the nuclear envelope.

Although hUba2 levels remained roughly constant, hAos1 levels increased during S phase and dropped in G₂ phase. The abundance of some SUMO-1-conjugated species similarly peaks in S phase (e.g., p160; Fig. 1A ), possibly suggesting that changes in hAos1 abundance influence the conjugation of these substrates. This mode of regulation would be interesting in light of evidence in budding and fission yeast linking SUMO-1 to checkpoint regulation of the cell cycle and mitotic progression. The changes in hAos1 levels during the cell cycle predict that some hUba2 should be in a hAos1-free form during G₂ phase. However, we found little evidence of hUba2- or hAos1-containing protein complexes outside of the hUba2/hAos1 heterodimer. The most likely solution to this apparent contradiction would be that such forms of hUba2 exist for a relatively short period during the cell cycle and thus were not obvious in asynchronous cell extracts. Pull-down assays with overexpressed epitope-tagged Uba2p in yeast suggested that Uba2p can interact with proteins in addition to Aos1p. It is possible that these associations were obvious because overexpression provided free pools of Uba2p.

Finally, we find that hUba2/hAos1 and hUbc9 alone are sufficient to recapitulate conjugation in vitro but are considerably less active than either HeLa cell (Fig. 2) or Xenopus egg extracts, suggesting that cellular extracts contain a stimulatory factor(s) that enhances the efficiency of conjugation. We do not believe that this stimulatory factor works through hUba2/hAos1, since additional purified E1 enzyme did not enhance SUMO-1 conjugation under these conditions. Because additional hUbc9 did increase conjugation, it is possible that the factor activates hUbc9. Alternatively, an uncharacterized E3-like ligase activity may promote SUMO-1 conjugation.

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram. Known steps in SUMO-1 conjugation are indicated in black; two potential sites of SUMO-1 pathway regulation are indicated in gray. See Conclusions for details.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0818fje ; to cite this article, use FASEB J. (June 18, 2001) 10.1096/fj.00-0818fje

² Present address: Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka 812-8582, Japan.

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