High Molecular Mass Protein Research Articles

Thiophosphorylation-induced Ca(2+) sensitization of guinea-pig ileum contractility is not mediated by Rho-associated kinase.

1. Incubation of beta-escin-permeabilized guinea-pig longitudinal ileal smooth muscle with ATP gamma S under conditions that do not lead to thiophosphorylation of regulatory light chains of myosin (r-MLC) increased subsequent Ca(2+) sensitivity of force and r-MLC phosphorylation. In this study we tested whether this is due to activation of the Rho and/or Rho-associated kinase (ROK) as it is the case in agonist-induced Ca(2+) sensitization. 2. The increase in Ca(2+) sensitivity induced by pretreatment with ATP gamma S at pCa > 8 with the myosin light chain kinase (MLCK) inhibitor ML-9 in rigor solution was associated with (35)S incorporation into the regulatory subunit of myosin light chain phosphatase (MLCP), MYPT1, and several other high molecular mass proteins. No thiophosphorylation of r-MLC, MLCK, caldesmon, calponin and CPI-17 was detected. 3. While the relatively specific inhibitor of ROK, Y 27632, inhibited the carbachol-induced increase in Ca(2+) sensitivity with an IC(50) of 1.4 microM, the ATP gamma S-induced increase in Ca(2+) sensitivity and thiophosphorylation of MYPT1 was not inhibited. Inhibiton of Rho by exoenzyme C3 also had no effect. 4. Only staurosporine (2 microM), but not the PKC inhibitor peptide 19-31, nor genistein nor PD 98059, inhibited the ATP gamma S-induced Ca(2+) sensitization of force, r-MLC phosphorylation, and the (35)S incorporation into MYPT1. 5. The staurosporine-sensitive kinase(s) appeared to be tightly associated with the contractile apparatus because treatment of Triton-skinned preparations with ATP gamma S also induced a staurosporine-sensitive increase in Ca(2+) sensitivity of contraction. Since there was very little immunoreactivity with antibodies to p(21)-associated kinase (PAK) in Triton-skinned preparations, the staurosporine-sensitive kinase most probably is not PAK. 6. GTP gamma S had an additive effect on ATP gamma S-induced sensitization at saturating concentrations of ATP gamma S. The additional effect of GTP gamma S was inhibited by Y 27632. 7. We conclude that treatment with ATP gamma S under ATP-free conditions, unmasks a staurosporine-sensitive kinase which induces a large increase in Ca(2+) sensitivity that is most likely to be due to thiophosphorylation of MYPT1. The kinase is distinct from ROK. The physiological significance of this kinase, which is tightly associated with the contractile apparatus, is not known at present.

Purification and Characterization of an Immunomodulatory Endometrial Protein, Glycodelin

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Evidence and characterization of a gene cluster required for the production of viscosin, a lipopeptide biosurfactant, by a strain of Pseudomonas fluorescens.

The genetic control of viscosin production was examined in a strain of Pseudomonas fluorescens (PfA7B) that causes broccoli head rot. Viscosin is a potent lipopeptide biosurfactant that enables the bacteria to come into intimate contact with the difficult-to-wet waxy heads of broccoli. Tn5 mutagenesis completely disrupted viscosin production as shown by HPLC analysis of the mutagenized cell lysates. The Vis- mutants retained their pectolytic capability and were able to decay potato slices. On broccoli, however, the Vis- mutants caused decay of wounded florets, but the decay failed to spread to adjacent nonwounded florets as had occurred with the wild-type PfA7B. Triparental matings of the Vis- mutants with their corresponding wild-type clones and the helper Escherichia coli HB101 carrying the mobilization plasmid pPK2013 resulted in three stable viscosin-producing transconjugants that caused typical decay of broccoli tissue. Linkage maps of clones and protein profiles showed that a 25-kb chromosomal DNA region of PfA7B affected the production of three high molecular mass proteins required for viscosin synthesis. These proteins, approximately 218, 215, and 137 kDa in size, likely compose a synthetase complex that assembles the nine amino acid peptide of viscosin and subsequently attaches this to the hydrophobic fatty acid component of the molecule. A probe made from this DNA region hybridized with DNA fragments of other phytopathogenic pseudomonads to varying degrees.

Oxidation of human lens recombinant αA-crystallin and cysteine-deficient mutants

Disulfide cross-linking, one of the results of oxidative stress, has been thought to play an important role in cataractogenesis. High molecular mass (HMM) protein aggregation also contributes to cataract development, and a prevailing speculation is that disulfide cross-linking induces HMM aggregation. However, there is no direct evidence to support this speculation. Dimerization is an effect of disulfide cross-linking but cannot explain the size of HMM aggregates observed in the lens. αA-crystallin has two cysteine residues (Cys131 and Cys142) and we have prepared three Cys-deficient mutants, two single mutants (C131I and C142I) and one double mutant (C131I/C142I). They were subjected to H 2O 2 oxidation in an ascorbate-FeCl 3-EDTA-H 2O 2 system. The effects of oxidation on the mutants, including changes in aggregate size and conformation, were compared with those of the wild-type αA-crystallin by FPLC gel filtration, absorption, fluorescence, and circular dichroism measurements. The results indicated that other amino acid residues besides Cys, such as Trp and Tyr, were also oxidized by H 2O 2. Disulfide dimerization alone seems to play a less important role in HMM aggregation than does the secondary conformational change resulting from the combined effect of the oxidation of Trp and Tyr as well as Cys.

PA-FABP, a novel marker of human epidermal transit amplifying cells revealed by 2D protein gel electrophoresis and cDNA array hybridisation

Human epidermal stem cells express higher levels of β1 integrins than their more differentiated daughters, transit amplifying cells. In a search for additional stem and transit cell markers we used proteomics and differential cDNA hybridisation to compare keratinocytes fractionated on the basis of β1 integrin expression. There were remarkably few differences between the two populations and none of the RNAs differed in abundance by more than 2-fold. Nevertheless, proteomics revealed upregulated expression of epidermal fatty acid binding protein (PA-FABP, also known as E-FABP), Annexin II and two keratin related proteins in the transit population. An unknown high molecular mass protein was upregulated in the stem cell population. The upregulation of PA-FABP was confirmed by Northern blotting and conventional and whole mount labelling of human epidermis. We conclude that PA-FABP is a novel marker of epidermal transit amplifying cells.

Solubilization, partial purification and photolabeling of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT).

In the present study, we defined experimental conditions that allowed the extraction of the integral membrane protein lysophospholipid:acyl-CoA acyltransferase (LAT, EC 2.3.1.23) from membranes while maintaining the full enzyme activity using the nonionic detergent n-octyl glucopyranoside (OGP) and solutions of high ionic strength. We found that the optimal OGP concentration depended on the ionic strength of the solubilization buffer. Fluorescence measurements with 1,6-diphenyl-1,3,5-hexatriene indicated that the critical micellar concentration (CMC) of OGP decreased with increasing salt concentrations. Analogous studies revealed that the zwitterionic detergent Chaps was ineffective in extracting LAT from membranes in the absence of salt, whereas its solubilization efficiency increased with increasing salt concentrations. Detailed lipid analysis of the different protein/lipid/detergent mixed micelles showed that the protein/lipid/OGP mixed micelles were relatively enriched with sphingomyelin (SPM) compared to protein/lipid/Chaps mixed micelles, indicating that the differences in the solubilization efficiency may be due to the ability to extract more SPM from membranes. When the protein/lipid/OGP mixed micelles were dissociated into protein/detergent and lipid/detergent complexes by the addition of increasing Chaps concentrations, one-tenth of the LAT enzyme activity was preserved making the enzyme accessible to protein purification. Analysis by native PAGE revealed that in the presence of excess Chaps a high molecular mass protein complex migrated into the gel which could be photolabeled by 125I-labelled-18-(4'-azido-2'-hydroxybenzoylamino)-oleyl-CoA. This fatty acid analogue has been shown to be a competitive inhibitor of LAT enzyme activity in the dark, and an irreversible inhibitor after photolysis. Therefore, this protein complex is assumed to contain the LAT enzyme.

Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements.

The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.

Effect of transglutaminase treatment on the functional properties of native and chymotrypsin-digested soy protein

Native and chymotrypsin-digested soy protein were polymerized by transglutaminase (TGase). SDS-PAGE pattern of the polymerized proteins showed that TGase treatment resulted in proteins of higher molecular mass above the stacking gel. The free amino groups of the polymerized proteins were greatly reduced. The solubility of the protein polymer was greatly improved at pH 2 and pH 8–12 whereas, at pH 4 and 6, it was least soluble. The solubility of the digest polymer was significantly improved at all pH levels, except pH 4. The protein polymer started to coagulate when the heating temperature exceeded 50°C. The digest polymer, on the other hand, resisted heat-induced aggregation up to 60°C; thereafter, its solubility declined slightly. The emulsifying and foaming properties of the digest polymer were greatly improved compared to the protein polymer. The protein polymer was observed to form a harder gel than the digest polymer.

Phylogenetic analysis of members of the genus Rickettsia using the gene encoding the outer-membrane protein rOmpB (ompB).

To confirm the phylogenetic analysis previously inferred by comparison of the citrate synthase and rOmpA gene sequences (gitA and ompA, respectively), the rOmpB gene (ompB) of 24 strains of the genus Rickettsia was amplified and sequenced. rOmpB is an outer-membrane protein of high molecular mass, the presence of which can be demonstrated in most rickettsiae by immunological cross-reactivity in Western blots. No PCR amplification was obtained with Rickettsia bellii or Rickettsia canadensis. For the other rickettsiae, phylogenetic analysis was inferred from the comparison of both the gene and derived protein sequences by using parsimony, maximum-likelihood and neighbour-joining methods which gave the same organization. All nodes were well supported (>86% bootstrap values), except in the cluster including Rickettsia africae strain S and Rickettsia parkeri, and this analysis confirmed the previously established phylogeny obtained from combining results from gltA and ompA. Based on phylogenetic data, the current classification of the genus Rickettsia is inappropriate, specifically its division into two groups, typhus and spotted fever. Integration of phenotypic, genotypic and phylogenetic data will contribute to the definition of a polyphasic taxonomy as has been done for other bacterial genera.

Evidence that ganglioside enriched domains are distinct from caveolae in MDCK II and human fibroblast cells in culture.

Cultures of MDCK II and human fibroblast cells were fed radioactive sphingosine and a radioactive GM3 ganglioside derivative containing a photoactivable group. The derived cell homogenates were treated with Triton X-100 and fractionated by sucrose-gradient centrifugation to prepare a detergent-insoluble membrane fraction known to be enriched in sphingolipid and caveolin-1, i.e. of caveolae. The detergent-insoluble membrane fraction prepared after feeding [1-3H]sphingosine to cells, was found to be highly enriched, with respect to protein content, in metabolically radiolabeled sphingomyelin and glycosphingolipids (about 18-fold). By feeding cells photoactivable radioactive GM3, after 2 h-chase, caveolin-1, CAV1, and proteins of high molecular mass became cross-linked to GM3, the cross-linking complexes being highly concentrated in the detergent-insoluble membrane fraction. The interaction between the ganglioside derivative and CAV1 was a time-dependent, transient process so that CAV1 cross-linking to GM3 was hardly detectable after a 24-h chase followed the pulse time. After a 24-h chase, only the high molecular mass proteins cross-linked to GM3 could be clearly observed. These results suggest that a portion of the GM3 administered to cells enters caveolae and moves to the glycosphingolipid domains, or enters caveolae that are then rapidly catabolized. Electron microscopy of cells in a culture immunostained with a monoclonal antibody to GM3 and a secondary gold-conjugated antibody detected several clusters of gangliosides on the plasma membranes separate from caveolae; gangliosides located inside the caveolae could not be detected. Scanning confocal microscopy of cells immunostained with anti-GM3 and anti-CAV1 Ig showed only a very small overlap with the CAV1 and GM3 signals. Thus, the biochemical and microscopic studies suggest that caveolae contain at most a low level of gangliosides and are separate from the GM3 ganglioside enriched domains.

Inactivation of phytotoxin produced by the rice sheath blight pathogen Rhizoctonia solani.

The rice sheath blight pathogen, Rhizoctonia solani, produces a toxin designated as RS-toxin, a carbohydrate compound containing mainly alpha-glucose and mannose. Different microflora were tested for RS-toxin inactivation. Isolates of Trichoderma viride inactivated this toxin when it was provided as the sole food source, and these isolates reduced the severity of toxin-induced symptoms and electrolyte leakage from rice cells. The best-performing isolate, TvMNT7, produced two extracellular proteins of 110 and 17 kDa. The high molecular mass protein was shown to have alpha-glucosidase activity. The purified 110 kDa protein was able to reduce RS-toxin activity.

Evidence for the presence of Pir-like proteins in Candida albicans

Pir proteins are unique proteins with internal repeat sequences that are reported to be present in the cell wall of Saccharomyces cerevisiae. They are covalently attached to the cell wall and can be released by mild alkali treatment. In this study the biotinylated cell wall preparations from Candida albicans and S. cerevisiae were extracted by alkali and β-1,3 glucanase and analyzed in parallel. Among the four bands detected by streptavidin, two proteins were recognized by the antibody to the S. cerevisiae Pir protein Hsp150. The antibody also detected a high molecular mass protein secreted in the growth medium of C. albicans. Using S. cerevisiae HSP150/ PIR2 gene as a probe, Southern and Northern hybridizations were performed with DNA and RNA of C. albicans. Hybridization with DNA digested with different restriction enzymes showed more than one hybridized fragment. An increased level of mRNA was found in heat shocked cells (37°C for 45 min compared to 25°C). Hybridization of ScHSP150 gene to mRNAs from cells grown in different media was also determined. Two transcripts of size approximately 3.5 kb and 2.0 kb were detected in mRNAs from cells grown in defined medium with glucose as carbon source or in the same medium supplemented with hemoglobin. The lower transcript of size 2.0 kb was absent in cells grown in medium with galactose as carbon source. A single band was also observed when cells were grown in rich medium. Together these results demonstrated the existence of β1,3 glucan linked proteins in C. albicans, which are related to Pir family proteins of S. cerevisiae.

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins.

A two-dimensional gel electrophoresis (2-DE) method that uses an agarose isoelectric focusing (IEF) gel in the first dimension (agarose 2-DE) was compared with an immobilized pH gradient 2-DE method (IPG-Dalt). The former method was shown to produce significant improvements in the 2-D electrophoretic separation of high molecular mass proteins larger than 150 kDa, up to 500 kDa, and to have a higher loading capacity, as much as 1.5 mg proteins in total for micropreparative runs. The extraction medium found best in this study for agarose 2-DE of mammal tissues was 6 M urea, 1 M thiourea, 0.5% 2-mercaptoethanol, protease inhibitor cocktail (Complete Mini EDTA-free), 1% Triton X-100 and 3% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Trichloroacetic acid (TCA) treatment of the agarose gel after IEF is to be carefully weighed beforehand, because some high molecular mass proteins were less likely to enter the second-dimensional polyacrylamide gel after TCA fixation, and proteins such as mouse skeletal muscle actin gave pseudospots in the agarose 2-DE patterns without TCA fixation. As a good compromise we suggest fixation of proteins in the agarose gel with TCA for one hour or less. The first-dimensional agarose IEF gel containing Pharmalyte as a carrier ampholyte was 180 mm in length and 2.5-4.8 mm in diameter. The gel diameter was shown to determine the loading capacity of the agarose 2-DE, and 1.5 mg liver proteins in total were successfully separated by the use of a 4.8 mm diameter agarose gel.

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor.

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

Preparative two-dimensional gel electrophoresis with agarose gels in the first dimension for high molecular mass proteins

Evidence for the presence of pir-like proteins in Candida albicans.

Pir proteins are unique proteins with internal repeat sequences that are reported to be present in the cell wall of Saccharomyces cerevisiae. They are covalently attached to the cell wall and can be released by mild alkali treatment. In this study the biotinylated cell wall preparations from Candida albicans and S. cerevisiae were extracted by alkali and beta-1,3 glucanase and analyzed in parallel. Among the four bands detected by streptavidin, two proteins were recognized by the antibody to the S. cerevisiae Pir protein Hsp150. The antibody also detected a high molecular mass protein secreted in the growth medium of C. albicans. Using S. cerevisiae HSP150/PIR2 gene as a probe, Southern and Northern hybridizations were performed with DNA and RNA of C. albicans. Hybridization with DNA digested with different restriction enzymes showed more than one hybridized fragment. An increased level of mRNA was found in heat shocked cells (37 degrees C for 45 min compared to 25 degrees C). Hybridization of ScHSP150 gene to mRNAs from cells grown in different media was also determined. Two transcripts of size approximately 3.5 kb and 2.0 kb were detected in mRNAs from cells grown in defined medium with glucose as carbon source or in the same medium supplemented with hemoglobin. The lower transcript of size 2.0 kb was absent in cells grown in medium with galactose as carbon source. A single band was also observed when cells were grown in rich medium. Together these results demonstrated the existence of beta1,3 glucan linked proteins in C. albicans, which are related to Pir family proteins of S. cerevisiae.

Association of Plectin with Z-Discs Is a Prerequisite for the Formation of the Intermyofibrillar Desmin Cytoskeleton

Plectin is a high-molecular mass protein (≈ 500 kd) that binds actin, intermediate filaments, and microtubules. Mutations of the plectin gene cause a generalized blistering skin disorder and muscular dystrophy. In adult muscle, plectin is colocalized with desmin at structures forming the intermyofibrillar scaffold and beneath the plasma membrane. To study the involvement of plectin in myofibrillogenesis, we analyzed the spatial and temporal expression patterns of plectin in cultured differentiating human skeletal muscle cells and its relationship to desmin intermediate filaments during this process. Northern and Western blot analyses demonstrated that at least two different plectin isoforms are expressed at all developmental stages from proliferating myoblasts to mature myotubes. Using immunocytochemistry, we show that the localization of plectin dramatically changes from a network-like distribution into a cross-striated distribution during maturation of myocytes. Double immunofluorescence experiments revealed that desmin and plectin are colocalized in premyofibrillar stages and in mature myotubes. Interestingly, plectin was often found to localize to the periphery of Z-discs during the actual alignment of neighboring myofibrils, and an obvious cross-striated plectin staining pattern was observed before desmin was localized in the Z-disc region. We conclude that the association of plectin with Z-discs is an early event in the lateral alignment of myofibrils that precedes the formation of the intermyofibrillar desmin cytoskeleton.

The chaperonin-related protein Tcm62p ensures mitochondrial gene expresssion under heat stress

Tcm62p, distantly related to chaperonins, is required for the assembly of succinate dehydrogenase in mitochondria of Saccharomyces cerevisiae and was proposed to exert chaperone activity. We demonstrate here crucial functions of Tcm62p under heat stress. It ensures mitochondrial gene expression at elevated temperatures and prevents heat-aggregation of the ribosomal subunit Var1p. Similar to chaperonins, Tcm62p forms a high molecular mass protein complex of approximately 850 kDa in the mitochondrial matrix space. These results suggest a more general chaperone function of Tcm62p in mitochondria.

Functional Expression of a Multisubstrate Deoxyribonucleoside Kinase from Drosophila melanogaster and Its C-terminal Deletion Mutants

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and Søndergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K(m) values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. Dm-dNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpes viral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The C-terminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.

Functional Heterogeneity of Small Ubiquitin-related Protein Modifiers SUMO-1 versus SUMO-2/3

Post-translational modification marked by the covalent attachment of the ubiquitin-like protein SUMO-1/SMT3C has been implicated in a wide variety of cellular processes. Recently, two cDNAs encoding proteins related to SUMO-1 have been identified in human and mouse. The functions and regulation of these proteins, known as SUMO-2/SMT3A and SUMO-3/SMT3B, remain largely uncharacterized. We describe herein quantitative and qualitative distinctions between SUMO-1 and SUMO-2/3 in vertebrate cells. Much of this was accomplished through the application of an antibody that recognizes SUMO-2 and -3, but not SUMO-1. This antibody detected multiple SUMO-2/3-modified proteins and revealed that, together, SUMO-2 and -3 constitute a greater percentage of total cellular protein modification than does SUMO-1. Intriguingly, we found that there was a large pool of free, non-conjugated SUMO-2/3 and that the conjugation of SUMO-2/3 to high molecular mass proteins was induced when the cells were subjected to protein-damaging stimuli such as acute temperature fluctuation. In addition, we demonstrated that SUMO-2/3 conjugated poorly, if at all, to a major SUMO-1 substrate, the Ran GTPase-activating protein RanGAP1. Together, these results support the concept of important distinctions between the SUMO-2/3 and SUMO-1 conjugation pathways and suggest a role for SUMO-2/3 in the cellular responses to environmental stress.