A vacuolar sorting receptor PV72 on the membrane of vesicles that accumulate precursors of seed storage proteins (PAC vesicles)

A novel vesicle, referred to as a precursor-accumulating (PAC) vesicle, mediates the transport of storage protein precursors to protein storage vacuoles in maturing pumpkin seeds. PV72, a type I integral membrane protein with three repeats of epidermal growth factor, was found on the membrane of the PAC vesicles. PV72 had an ability to bind to pro2S albumin, a storage protein precursor, in a Ca(2+)-dependent manner, via the C-terminal region of pro2S albumin, which was found to function as a vacuolar targeting signal. This implies that PV72 is a vacuolar sorting receptor of the storage protein. PV72 was specifically and transiently accumulated at the middle stage of seed maturation in association with the synthesis of storage proteins. Subcellular fractionation showed that PV72 was also accumulated in the microsomal fraction. A fusion protein consisting of GFP and the transmembrane domain and the cytosolic tail of PV72 was localized in Golgi complex. PV72 in the isolated PAC vesicles had a complex type of oligosaccharide, indicating that PV72 passed though the Golgi complex. These results suggest that PV72 is recycled between PAC vesicles and Golgi complex/post-Golgi compartments. PV72 appears to be responsible for recruiting pro2S albumin molecules from the Golgi complex to the PAC vesicles.     

Accumulation of a fusion protein containing 2S albumin induces novel vesicles in vegetative cells of Arabidopsis.

We have previously reported that precursor-accumulating (PAC) vesicles found exclusively in developing seeds are involved in a transport of seed storage proteins, such as 2S albumin, from the endoplasmic reticulum to protein-storage vacuoles. Here, we constructed chimeric genes that encode fusion proteins consisting of both various lengths of polypeptides derived from pumpkin 2S albumin and a selectable marker enzyme, phosphinothricin acetyltransferase. The chimeric genes were expressed in transgenic Arabidopsis in order to investigate the mechanism of the PAC vesicle formation. A fusion protein expressed by one of the chimeric genes is accumulated as a proprotein-precursor form, and localized in novel vesicles of vegetative cells. The vesicles show distinct features that well much to the PAC vesicles. Despite of the accumulation of the fusion protein, the transgenic Arabidopsis is still sensitive to phosphinothricin. Phosphinothricin acetyltransferase contained in the fusion protein is obviously compartmentalized in the PAC-like vesicles that do not permit the detoxification of this herbicide. These results indicate that the PAC-like vesicle can be induced in vegetative cells by the ectopic expression of the protein that is destined to be compartmentalized into the PAC vesicles.     

Calcium-mediated association of a putative vacuolar sorting receptor PV72 with a propeptide of 2S albumin.

PV72, a type I membrane protein with three epidermal-growth factor (EGF)-like motifs, was found to be localized on the membranes of the precursor-accumulating (PAC) vesicles that accumulated precursors of various seed storage proteins. To clarify the function of PV72 as a sorting receptor, we expressed four modified PV72s and analyzed their ability to bind the internal propeptide (the 2S-I peptide) of pro2S albumin by affinity chromatography and surface plasmon resonance. The recombinant PV72 specifically bound to the 2S-I peptide with a K(D) value of 0.2 microm, which was low enough for it to function as a receptor. The EGF-like motifs modulated the Ca(2+)-dependent conformational change of PV72 to form a functional pocket for the ligand binding. The binding of Ca(2+) stabilizes the receptor-ligand complex even at pH 4.0. The association and dissociation of PV72 with the ligand is modulated by the Ca(2+) concentration (EC(50) value = 40 microm) rather than the environmental pH. Overall results suggest that Ca(2+) regulates the vacuolar sorting mechanism in higher plants.     

Vesicle transport and processing of the precursor to 2S albumin in pumpkin

Cell fractionation of pulse-chase-labeled developing pumpkin cotyledons demonstrated that proprotein precursor to 2S albumin is transported from the endoplasmic reticulum to dense vesicles and then to the vacuoles, in which pro2S albumin is processed to the mature 2S albumin. Immunocytochemical analysis showed that dense vesicles of about 300 nm in diameter mediate the transport of pro2S albumin to the vacuoles.
The primary structure of the precursor (16 578 Da) to pumpkin 2S albumin has been deduced from the nucleotide sequence of an isolated cDNA insert. The presence of a hydrophobic signal peptide at the N-terminus indicates that the precursor is a preproprotein that is converted into pro2S albumin after cleavage of the signal peptide. N-terminal sequencing of the pro2S albumin in the isolated vesicles revealed that the signal peptide is cleaved off co-translationally on the C-terminal side of alanine residue 22 of prepro2S albumin. By contrast, post-translational cleavages occur on the C-terminal sides of asparagine residues 35 and 74, which are conserved among precursors to 2S albumin from different plants. Hydropathy analysis revealed that the two asparagine residues are located in the hydrophilic regions of pro2S albumin. These findings suggest that a vacuolar processing enzyme can recognize exposed asparagine residues on the molecular surface of pro2S albumin and cleave the peptide bond on the C-terminal side of each asparagine residue to produce mature 2S albumin in the vacuoles.     

Transport of storage proteins to protein storage vacuoles is mediated by large precursor-accumulating vesicles

Novel vesicles that accumulate large amounts of proprotein precursors of storage proteins were purified from maturing pumpkin seeds. These vesicles were designated precursor-accumulating (PAC) vesicles and had diameters of 200 to 400 nm. They contained an electron-dense core of storage proteins surrounded by an electron-translucent layer, and some vesicles also contained small vesicle-like structures. Immunocytochemical analysis revealed numerous electron-dense aggregates of storage proteins within the endoplasmic reticulum. It is likely that these aggregates develop into the electron-dense cores of the PAC vesicles and then leave the endoplasmic reticulum. Immunocytochemical analysis also showed that complex glycans are associated with the peripheral region of PAC vesicles but not the electron-dense cores, indicating that Golgi-derived glycoproteins are incorporated into the PAC vesicles. These results suggest that the unique PAC vesicles might mediate a transport pathway for insoluble aggregates of storage proteins directly to protein storage vacuoles.     

Transport of ricin and 2S albumin precursors to the storage vacuoles of Ricinus communis endosperm involves the Golgi and VSR-like receptors

We have studied the transport of proricin and pro2S albumin to the protein storage vacuoles of developing castor bean (Ricinus communis L.) endosperm. Immunoelectron microscopy and cell fractionation reveal that both proteins travel through the Golgi apparatus and co-localize throughout their route to the storage vacuole. En route to the PSV, the proteins co-localize in large (>200 nm) vesicles, which are likely to represent developing storage vacuoles. We further show that the sequence-specific vacuolar sorting signals of both proricin and pro2SA bind in vitro to proteins that have high sequence similarity to members of the VSR/AtELP/BP-80 vacuolar sorting receptor family, generally associated with clathrin-mediated traffic to the lytic vacuole. The implications of these findings in relation to the current model for protein sorting to storage vacuoles are discussed.     

Characterization of calpain I-binding proteins in human erythrocyte plasma membrane

The calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2(+)-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5 microM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100 micrograms/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37 degrees C, although the binding to the vesicles pretreated with 200 micrograms/ml of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblotting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i.e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).     

Characterization of organelles in the vacuolar-sorting pathway by visualization with GFP in tobacco BY-2 cells

We have shown the localization and mobilization of modified green fluorescent proteins (GFPs) with various signals in different compartments in a vacuolar-sorting system of tobacco BY-2 cells. In contrast to the efficient secretion of GFP from the transformed cells expressing SP-GFP composed of a signal peptide and GFP, accumulation of GFP in the vacuoles was observed in the cells expressing SP-GFP fused with the C-terminal peptide of pumpkin 2S albumin. This indicated that this peptide is sufficient for vacuolar targeting. Interestingly, the fluorescence in the vacuoles disappeared sharply at 7 d after inoculation of the cells, but it appeared again after re-inoculation into a new culture medium. When SP-GFP was fused with the region, termed PV72C, including a transmembrane domain and a cytosolic tail of a vacuolar-sorting receptor PV72, GFP-PV72C was detected in the Golgi-complex-like small particles. Prolonged culture showed that GFP-PV72C that reached the prevacuolar compartments was cleaved off the PV72C region to produce GFP, that arrived at the vacuoles to be diffused. These findings suggested that the vacuolar-sorting receptor might be recycled between the Golgi complex and prevacuolar compartments.     

Analysis of picornavirus 2A(pro) proteins: separation of proteinase from translation and replication functions.

The poliovirus (PV) genome was manipulated by replacing its 2A-encoding sequence with the corresponding sequence of coxsackie B4 virus (CBV4) or human rhinovirus type 2 (HRV2). In vitro translation of the resulting chimeric PV genomes revealed a normal cis-cleavage activity for both heterologous 2A(pro) proteinases in the chimeric PV polyproteins. However, only the genome containing the 2A-encoding sequence of CBV4 (PV/CBV4-2A) yielded viable virus in transfected cells, producing a mixture of large and small plaques on HeLa cell monolayers. The large-plaque variants were found to contain single-amino-acid mutations at a specific site near the C terminus of the CBV4 2A(pro) protein. When the same single-amino-acid mutations were directly introduced into the parental PV/CBV4-2A genome, chimeric viruses with a large-plaque phenotype and a wild-type PV-like growth pattern were obtained upon transfection, an observation demonstrating that these point mutations alone had a drastic effect on the growth of the PV/CBV4 chimeric virus. On the other hand, the chimeric genome containing the 2A-encoding sequence of HRV2 (PV/HRV2-2A) produced a null phenotype in transfected HeLa cells, although low-level replication of this chimeric genome was evident. We conclude that only 2A(pro) of the more closely related enterovirus CBV4 is able to functionally substitute for that of PV in vivo, and a subtle genetic modification of the CBV4 2A(pro) protein results in a drastic improvement in the growth of the chimeric PV/CBV4-2A virus. In addition, this chimeric cDNA approach enabled us to dissect multiple biological functions encoded by the 2A(pro) proteins.     

Characteristics and significance of albumin-positive hepatocytes in analbuminemic rats

Analbuminemic rats (NAR) are a mutant strain in which splicing of the albumin mRNA is blocked due to a seven-base-pair deletion in an intron of the albumin gene. NAR liver contains a few hepatocytes that react with anti-rat albumin antibody (Alb+ hepatocytes), and these cells increase in number during aging and on treatment with hepatocarcinogens. To characterize these Alb+ hepatocytes, we examine their albumin mRNA, the biochemical specificity of their albumin, and its intracellular distribution. Signals of albumin mRNA were observed in a few hepatocytes by in situ hybridization. Moreover, a small amount of cytoplasmic albumin mRNA was detected by RNA blot analysis in the liver of aged NAR and NAR treated with 3'-methyl-4-diaminoazobenzene (DAB). Immunoelectron microscopic examination revealed the cisternae of the rough and smooth endoplasmic reticula, Golgi complexes, and secretory vesicles of the Alb+ hepatocyte of NAR being filled with material that reacted with anti-rat albumin antibody. These facts suggested that albumin was gradually synthesized in Alb+ hepatocytes but that its secretion was disturbed. The albumin-like proteins of NAR were shown by Western blot analysis to consist of three species of 68 kDa, 50 kDa, and 25 kDa proteins. The 50 kDa albumin was thought to be formed by exon-skipping splicing of the albumin mRNA precursor, which was recently reported by Shalaby and Shafritz (Proc. Natl. Acad. Sci. USA 87, 2652-2656 (1990)). The 25 kDa protein was suspected to be formed by fragmentation of the 50 kDa protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

'Detergent-like' permeabilization of anionic lipid vesicles by melittin

Melittin (MLT), the 26-residue toxic peptide from the European honeybee Apis mellifera, is widely used for studying the principles of membrane permeabilization by antimicrobial and other host-defense peptides. A striking property of MLT is that its ability to permeabilize zwitterionic phospholipid vesicles is dramatically reduced upon the addition of anionic lipids. Because the mechanism of permeabilization may be fundamentally different for the two types of lipids, we examined MLT-induced release of entrapped fluorescent dextran markers of two different molecular masses (4 and 50 kDa) from anionic palmitoyloleoylphosphatidylglycerol (POPG) vesicles. Unlike release from palmitoyloleoylphosphatidylcholine (POPC) vesicles, which is highly selective for the 4 kDa marker, implying release through pores of about 25 A diameter [Ladokhin et al., Biophys. J. 72 (1997) 1762], release from POPG vesicles was found to be non-selective, i.e., 'detergent-like'. Oriented circular dichroism measurements of MLT in oriented POPG and POPC multilayers disclosed that alpha-helical MLT can be induced to adopt a transbilayer orientation in POPC multilayers, but not in POPG multilayers. The apparent inhibition of MLT permeabilization by anionic membranes may thus be due to suppression of translocation ability.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

m-Calpain-mediated cleavage of Na+/Ca2+ exchanger-1 in caveolae vesicles isolated from pulmonary artery smooth muscle

Using m-calpain antibody, we have identified two major bands corresponding to the 80 kDa large and the 28 kDa small subunit of m-calpain in caveolae vesicles isolated from bovine pulmonary artery smooth muscle plasma membrane. In addition, 78, 35, and 18 kDa immunoreactive bands of m-calpain have also been detected. Casein zymogram studies also revealed the presence of m-calpain in the caveolae vesicles. We have also identified Na⁺/Ca²⁺ exchanger-1 (NCX1) in the caveolae vesicles. Purification and N-terminal sequence analyses of these two proteins confirmed their identities as m-calpain and NCX1, respectively. We further sought to determine the role of m-calpain on calcium-dependent proteolytic cleavage of NCX1 in the caveolae vesicles. Treatment of the caveolae vesicles with the calcium ionophore, A23187 (1 μM) in presence of CaCl₂ (1 mM) appears to cleave NCX1 (120 kDa) to an 82 kDa fragment as revealed by immunoblot study using NCX1 monoclonal antibody; while pretreatment with the calpain inhibitors, calpeptin or MDL28170; or the Ca²⁺ chelator, BAPTA-AM did not cause a discernible change in the NCX protein profile. In vitro cleavage of the purified NCX1 by the purified m-calpain supports this finding. The cleavage of NCX1 by m-calpain in the caveolae vesicles may be interpreted as an important mechanism of Ca²⁺ overload, which could arise due to inhibition of Ca²⁺ efflux by the forward-mode NCX and that could lead to sustained Ca²⁺ overload in the smooth muscle leading to pulmonary hypertension.     

A novel membrane protein that is transported to protein storage vacuoles via precursor-accumulating vesicles

A novel protein, MP73, was specifically found on the membrane of protein storage vacuoles of pumpkin seed. MP73 appeared during seed maturation and disappeared rapidly after seed germination, in association with the morphological changes of the protein storage vacuoles. The MP73 precursor deduced from the isolated cDNA was composed of a signal peptide, a 24-kD domain (P24), and the MP73 domain with a putative long alpha-helix of 13 repeats that are rich in glutamic acid and arginine residues. Immunocytochemistry and immunoblot analysis showed that the precursor-accumulating (PAC) vesicles (endoplasmic reticulum-derived vesicles responsible for the transport of storage proteins) accumulated proMP73, but not MP73, on the membranes. Subcellular fractionation of the pulse-labeled maturing seed demonstrated that the proMP73 form with N-linked oligosaccharides was synthesized on the endoplasmic reticulum and then transported to the protein storage vacuoles via PAC vesicles. Tunicamycin treatment of the seed resulted in the efficient deposition of proMP73 lacking the oligosaccharides (proMP73 Delta Psi) into the PAC vesicles but no accumulation of MP73 in vacuoles. Tunicamycin might impede the transport of proMP73 Delta Psi from the PAC vesicles to the vacuoles or might make the unglycosylated protein unstable in the vacuoles. After arrival at protein storage vacuoles, proMP73 was cleaved by the action of a vacuolar enzyme to form a 100-kD complex on the vacuolar membranes. These results suggest that PAC vesicles might mediate the delivery of not only storage proteins but also membrane proteins of the vacuoles.     

An ER-localized form of PV72, a seed-specific vacuolar sorting receptor, interferes the transport of an NPIR-containing proteinase in Arabidopsis leaves

Putative vacuolar sorting receptors that bind to the vacuolar targeting signals have been found in various plants; pumpkin PV72, pea BP-80 and Arabidopsis AtELP. PV72 is a seed-specific receptor that is predicted to sort seed storage proteins to protein storage vacuoles. Analysis by surface plasmon resonance showed that the lumenal domain of PV72 bound to an NPIR (a typical vacuolar targeting signal)-containing peptide of the precursor of a cysteine proteinase, AtALEU, in the presence of Ca(2+) (K(D) = 0.1 micro M). To elucidate the receptor-dependent transport of vacuolar proteins in plant cells, we produced transgenic Arabidopsis plants that expressed a fusion protein (PV72-HDEL) composed of the lumenal domain of PV72 and an endoplasmic reticulum (ER)-retention signal, HDEL. The expression of PV72-HDEL induced the accumulation of the AtALEU precursor. The accumulation level of the AtALEU precursor was dependent on that of PV72-HDEL. In contrast, it did not induce the accumulation of a precursor of another cysteine proteinase, RD21, which contains no NPIR. Detailed subcellular localization revealed that both the AtALEU precursor and PV72-HDEL accumulated in the ER fraction. We found that most of the AtALEU precursor molecules formed a complex with PV72-HDEL. The AtALEU precursor might be trapped by PV72-HDEL in the ER and not transported to the vacuoles. This in-planta analysis supports the hypothesis that an Arabidopsis homolog of PV72 functions as a sorting receptor for the NPIR-containing proteinase. The overall results suggest that vacuolar sorting receptors for the protein storage vacuoles and the lytic vacuoles share the similar recognition mechanism for a vacuolar targeting signal.     

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.     

ADP ribosylation factor and a 14-kD polypeptide are associated with heparan sulfate-carrying post-trans-Golgi network secretory vesicles in rat hepatocytes

Constitutive secretory vesicles carrying heparan sulfate proteoglycan (HSPG) were identified in isolated rat hepatocytes by pulse-chase experiments with [35S]sulfate and purified by velocity-controlled sucrose gradient centrifugation followed by equilibrium density centrifugation in Nycodenz. Using this procedure, the vesicles were separated from plasma membranes, Golgi, trans-Golgi network (TGN), ER, endosomes, lysosomes, transcytotic vesicles, and mitochondria. The diameter of these vesicles was approximately 100-200 nm as determined by electron microscopy. A typical coat structure as described for intra- Golgi transport vesicles or clathrin-coated vesicles could not be seen, and the vesicles were not associated with the coat protein beta-COP. Furthermore, the vesicles appear to represent a low density compartment (1.05-1.06 g/ml). Other constitutively secreted proteins (rat serum albumin, apolipoprotein E, and fibrinogen) could not be detected in purified HSPG-carrying vesicles, but banded in the denser fractions of the Nycodenz gradient. Moreover, during pulse-chase labeling with [35S]methionine, labeled albumin did not appear in the post-TGN vesicle fraction carrying HSPGs. These findings indicate sorting of HSPGs and albumin into different types of constitutive secretory vesicles in hepatocytes. Two proteins were found to be tightly associated with the membranes of the HSPG carrying vesicles: a member of the ADP ribosylation factor family of small guanine nucleotide-binding proteins and an unknown 14-kD peripheral membrane protein (VAPP14). Concerning the secretory pathway, we conclude from these results that ADP ribosylation factor proteins are not only involved in vesicular transport from the ER via the Golgi to the TGN, but also in vesicular transport from the TGN to the plasma membrane.     

The Ricinus communis 2S albumin precursor: a single preproprotein may be processed into two different heterodimeric storage proteins

Summary TheRicinus communis (castor bean) 2S albumin is a heterodimer of glutamine-rich, disulphidelinked 4 and 7 kDa polypeptides. A cDNA library was constructed using mRNA from maturing castor bean endosperm as template. Clones containing sequences complementary to albumin mRNA were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing sequences predicted for a peptide present in the 2 S albumin large subunit. The nucleotide sequence contained an open reading frame encoding a preproprotein of 258 amino acid residues. The preproprotein included both polypeptides of the previously sequenced 2S albumin. In addition, this precursor included two further glutamine-rich sequences which, in terms of their size and conserved cysteine residues typically found in seed proteins of the 2S albumin superfamily, possibly represent the small and large polypeptide subunits of a second heterodimeric storage protein. A post-translational processing scheme is proposed which would result in a single preproprotein generating two distinct heterodimeric 2S albumins. The generation of a second heterodimer seems likely since polypeptide candidates for its small and large subunits were found in theRicinus 2S albumin fraction, and N-terminal protein sequencing confirmed the existence of the putative small subunit.     

SUMO-1 modification of human cytomegalovirus IE1/IE72

Human cytomegalovirus (HCMV) immediate-early protein IE1/IE72 is involved in undermining many cellular processes including cell cycle regulation, apoptosis, nuclear architecture, and gene expression. The multifunctional nature of IE72 suggests that posttranslational modifications may modulate its activities. IE72 is a phosphoprotein and has intrinsic kinase activity (S. Pajovic, E. L. Wong, A. R. Black, and J. C. Azizkhan, Mol. Cell. Biol. 17:6459-6464, 1997). We now demonstrate that IE72 is covalently conjugated to the small ubiquitin-like modifier (SUMO-1). SUMO-1 is an 11.5-kDa protein that is conjugated to multiple proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. A covalently modified protein migrating at approximately 92 kDa, which is stabilized by a SUMO-1 hydrolase inhibitor, is revealed by Western blotting with anti-IE72 of lysates from cells infected with HCMV or cells expressing IE72. SUMO modification of IE72 was confirmed by immunoprecipitation with anti-IE72 and anti-SUMO-1 followed by Western blotting with anti-SUMO-1 and anti-IE72, respectively. Lysine 450 is within a sumoylation consensus site (I,V,L)KXE; changing lysine 450 to arginine by point mutation abolishes SUMO-1 modification of IE72. Inhibition of protein phosphatase 1 and 2A, which increases the phosphorylation of IE72, suppresses the formation of SUMO-1-IE72 conjugates. Both wild-type IE72 and IE72(K450R) localize to nuclear PML oncogenic domains and disrupt them. Studies of protein stability, transactivation, and complementation of IE72-deficient HCMV (CR208) have revealed no significant differences between wild-type IE72 and IE72(K450R).