Immunological Studies of a 21 kDa Cellular Component Efficiently Incorporated into Rabies Virion Grown in a BHK-21 Cell Culture

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion-associated 21 kDa polypeptide (referred to as VAP21) from a BHK-21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.     

Further characterization of a CD99-related 21-kDa transmembrane protein (VAP21) expressed in Syrian hamster cells and its possible involvement in vesicular stomatitis virus production

The VAP21, a CD99-related 21-kDa transmembrane protein, was first detected in the enveloped virions that were grown in a Syrian hamster-derived cell line, BHK-21 (Sagara et al., 1997; Yamamoto et al., 1999). We further tried to elucidate the nature and properties of VAP21. The VAP21 was detected in various organs of the Syrian hamster as well as in the Syrian hamster-derived cell lines (BHK-21 and HmLu-1). We could not detect the VAP21 antigen in other cell lines derived from other animal species we examined, including a Chinese hamster (CHO-K1), mouse (neuroblastoma C1300, clone NA), dog (MDCK), monkey (COS-7), and human (HeLa, HepG2). We tried to introduce the VAP21 gene into VAP21-negative cell lines using a tetracycline-regulated gene expression system. All of our trials, however, resulted in failure to establish stably positive inducible cell lines. To the contrary, we could easily establish the VAP21-overexpressing cell lines from the Syrian hamster cell lines, which were successfully grown and maintained without any loss of VAP21 expression even under the induced culture conditions. In such VAP21-overexpressing cells, production of the vesicular stomatitis virus (VSV) was increased several-fold, while suppression of the VAP21 expression resulted in reducing the VSV yields. From these results, we conclude that the VAP21 is a physiologically active cell membrane component of some animal species including the Syrian hamster, and might positively be involved in the VSV replication.     

The rabies virion-associated 100-kDa polypeptide (VAP100) is a host-derived minor component of the viral envelope

We investigated a minor polypeptide component of 100-kDa detected in the rabies virion (referred to as VAP100) by using a monoclonal antibody (mAb), #16743, which was shown to recognize the SDS-denatured VAP100 antigen by immunoblot analyses. Although the VAP100 antigen was hardly detectable in the cell by usual immunoblot methods with this mAb, we could detect the antigen by a luminescent immunoblot method as well as by immunoprecipitation from the metabolically radiolabeled cell lysates and virions. Fluorescent antibody (FA) staining with mAb #16743 detected the uniformly distributed antigen on the formalin-fixed normal BHK-21 cells, while slight accumulation of the antigen was also seen in the Golgi area when the cells were permeabilized by treatment with Triton X-100 after fixation. Rabies virus infection induced alteration of the behavior of VAP100 to show a spotted distribution pattern in virus-infected cells. Double FA staining with mAb #16743 and rabbit antibody against the rabies virus envelope antigen demonstrated colocalized distribution of the viral envelope antigens and VAP100 in the cell. From these results, we think that VAP100 is a membrane-associated component of the cell, and its colocalized distribution with the viral envelope antigens in the cell implicates an intimate association of the VAP100 with viral envelope protein(s) and a reflection of possible involvement in the efficient incorporation of VAP100 into the virion.     

Association of a cellular 21-kDa transmembrane protein (VAP21) with enveloped viruses.

We reported previously that the rabies virions contained a 21-kDa cellular transmembrane protein (referred to as VAP21) as a minor component (Sagara, J. et al, Microbiol. Immunol. 41(12): 947-955, 1997). In this study, we further examined the possible interactions of VAP21 with other enveloped viruses, including the vesicular stomatitis virus (VSV; negative-stranded RNA virus), Sindbis virus (positive-stranded RNA virus) and herpes simplex virus type 1 (HSV-1; double-stranded DNA virus). An immunoblot analysis demonstrated that all of these enveloped viruses contained VAP21 in the virion as a minor component. Immunoprecipitation studies suggested that VAP21 was associated with certain viral proteins in the cell, such as the matrix (M) protein of VSV, a capsid protein of Sindbis virus, and at least a capsid protein (VP5) of HSV-1. The association was disrupted by treatment with 0.5% sodium dodecyl sulfate, but resistant to the treatment with 1% NP-40 plus 1% deoxycholate. These results suggest that: 1) VAP21 is not primarily associated with the viral transmembrane glycoprotein but rather with the internal viral protein, and, 2) this association would cause the efficient incorporation of VAP21 into the virion.     

Isolation and replication of rabies virus in C6 rat glioma cells (clone CCL-107).

The susceptibility of the C6 rat glioma cell line (ATCC; CCL-107) to rabies virus was characterized. The kinetics of infection performed with a fixed and a wild strain (from an infected cow) of rabies virus was monitored by direct immunofluorescence. Fluorescent cytoplasmic bodies were readily observed by UV microscopy from 24 hours post-infection (hpi) onwards. The ability of C6 to produce rabies infective virion particles was confirmed by determining the viral titres present in the supernatants of infected cultures, by both BHK-21 cell infection and mice inoculation. C6 cells produced similar viral titres to those produced by BHK-21 for both strains used. In addition, the yield of rabies glycoprotein was assessed by ELISA. In general, BHK-21 and C6 cells infected either by PV or with the wild rabies strain produced similar amounts of rabies glycoprotein. At 96 hpi, however, when the glycoprotein production peaked, BHK-21 infected with the wild strain produced significantly higher amounts of glycoprotein than C6. Subsequently, the optimal conditions for isolation of wild rabies virus strains from C6 cells were established and these proved to be as sensitive as NA cells in detecting 10 wild rabies samples. Due to the high sensitivity exhibited, C6 rat glioma cells present a new and useful system for rabies virus investigation.     

ERp99, an abundant, conserved glycoprotein of the endoplasmic reticulum, is homologous to the 90-kDa heat shock protein (hsp90) and the 94-kDa glucose regulated protein (GRP94)

We have isolated an expressible full-length cDNA clone encoding murine ERp99, an abundant, conserved transmembrane glycoprotein of the endoplasmic reticulum membrane. ERp99 is synthesized as a 92,475-kDa precursor containing 802 amino acids. It possesses a signal peptide of 21 amino acids which is cleaved cotranslationally. Analysis of the amino acid sequence deduced from the nucleotide sequence of the cDNA clone led us to propose a model for the orientation of ERp99 in the endoplasmic reticulum membrane. In this model, ERp99 possesses one membrane-spanning, stop transfer segment in the N-terminal region. The protein chain passes through the membrane only once, and approximately 75% of the protein remains on the cytoplasmic side of the ER membrane. Comparison of the ERp99 sequence to the sequence of other proteins revealed that ERp99 has extensive homology with the 90-kDa heat shock protein of Saccharomyces cerevisiae (hsp90) and the 83-kDa heat shock protein of Drosophila melanogaster. In addition, the N terminus of mature ERp99 is identical to that of the 94-kDa glucose regulated protein (GRP94) of mammalian cells.     

Studies on the Rabies Virus RNA Polymerase: 2. Possible Relationships between the Two Forms of the Non-Catalytic Subunit (P Protein)

We investigated the relationship between the two forms of rabies virus P protein, a non-catalytic subunit of rabies virus RNA polymerase. The two displayed different electrophoretic mobilities as 37- and 40-kDa polypeptides, hence termed as p37 and p40, respectively. Double labeling experiments with [³H]leucine and [³²P]orthophosphate demonstrated that p40 was much more phosphorylated than p37. Treatment of the virion proteins with alkaline phosphatase eliminated only p40, and not 37-kDa polypeptide. The p37 was a major product of the P gene, and was accumulated in the infected cell and incorporated into the virion. On the other hand, p40 was apparently detected only in the virion, and little detected in the cells. Treatment of infected cells with okadaic acid, however, resulted in significant accumulation of p40 in the cell, suggesting that p40 was continuously produced in the cell but dephosphorylated quickly. We detected both 37- and 40-kDa products in P cDNA-transfected animal cells, while only a 37-kDa product was produced in Escherichia coli. Incubation of 37-kDa products from E. coli with the lysates of animal cells in vitro resulted in the production of a 40-kDa product, which was also shown to be suppressed by the heparin. From these results, it is suggested that p40 is produced by the hyperphosphorylation of a 37-kDa polypeptide, which depends on certain heparin-sensitive cellular enzyme(s) and occurs even in the absence of the other viral gene products, and that p40 is reverted quickly to p37 in the infected cells, probably being dependent on some virus-induced factor(s).     

Ineffectiveness and comparative pathogenicity of attenuated rabies virus vaccines for the striped skunk (Mephitis mephitis)

Three attenuated rabies virus vaccines (SAD-B19, ERA/BHK-21, AZA 2) were compared for efficacy and safety in the striped skunk (Mephitis mephitis) by the oral and intranasal routes. The SAD-B19 and ERA/BHK-21 vaccines were given orally; all three vaccines were given intranasally. Oral administration of SAD-B19 and ERA/BHK-21 vaccines induced neither seroconversion nor significant protection against rabies challenge. One skunk which consumed a SAD-B19 vaccine-laden bait succumbed to vaccine-induced rabies. Intranasal instillation of the three vaccines resulted in the deaths of two of six (AZA 2), three of six (ERA/BHK-21) and six of six (SAD-B19) skunks.     

Lipids of rabies virus and BHK-21 cell membranes.

The lipid composition of highly purified Flury strain of rabies virus (HEP) propagated in BHK-21 cells in a chemically defined medium was observed to be 6.7% neutral lipids, 15.8% phospholipids, and 1.5% glycolipids. In the virion, phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin were the most abundant phospholipids, accounting for 90% of the total, and the molar ratio of cholesterol to phospholipid was 0.48. Uninfected BHK-21 cell membranes were obtained by nitrogen cavitation techniques and separated by density gradient centrifugation, and the membranes were assayed for purity using 5'-nucleotidase, cytochrome oxidase, and reduced nicotinamide adenine dinucleotide phosphate diaphorase activities. Lipids of the plasma membrane were enriched in cholesterol, phosphatidylcholine, and phosphatidylethanolamine. In contrast, membranes of the endoplasmic reticulum were enriched in phosphatidylcholine, but contained smaller amounts of phosphatidylethanolamine and sphingomyelin. Comparison of the fatty acyl chains of virus and membranes from uninfected cells revealed the virion to have the lowest ratio of C18:1 to C18:0 (1.771), compared with values of about 3.0 for the plasma membrane and endoplasmic reticulum. Total polyenoic fatty acids were enriched in the plasma membrane, whereas the virus contained higher amounts of total saturates than either of the two membrane preparations. Analysis of the polar and neutral lipid fractions as well as the acyl chain analysis suggests the virion has a lipid composition that is intermiediate to that of the plasma membrane and endoplasmic reticulum and is consistent with the view that numerous viral particles are synthesized de novo by not utilizing a preexisting membrane template. From the ratio of cholesterol to phospholipid of 0.48, we calculated that 1.92 X 10(5) molecules of lipid would cover 4.14 X 10(4) nm2 in the form of a bilayer. Considerations of the molecular dimensions of the rabies envelope (total surface area, 5 X 10(4) nm2) as a bilayer suggest that some penetration of lipids by envelope proteins (M and G) is necessary.     

Characterization of a slow-migrating component of the rabies virus matrix protein strongly associated with the viral glycoprotein

We investigated multiple forms of rabies virus matrix (M) protein. Under non-reducing electrophoretic conditions, we detected, in addition to major bands of monomer forms (23- and 24-kDa) of M protein, an M antigen-positive slow-migrating minor band (about 54 kDa) in both the virion and infected cells. Relative contents of the 54-kDa and monomer components in the virion were about 20-30% and 70-80% of the whole M protein, respectively, while the content of the 54-kDa component was smaller (about 10-20% of the total M protein) in the cell than in the virion. The 54-kDa components could be extracted from the infected cells with sodium deoxycholate, but they were quite resistant to extraction with 1% nonionic detergents by which most monomer components were solubilized. The 54-kDa component was precipitated more efficiently than the monomer by a monoclonal antibody (mAb; #3-9-16), which recognized a linear epitope located at the N-terminal of the M protein. The mAb #3-9-16 coprecipitated the viral glycoprotein (G), which was demonstrated to be due to strong association between the G and 54-kDa component of the M protein. Monomers and the 54-kDa polypeptide migrated to the same isoelectric point (pI) in twodimensional (2-D) gel electrophoresis, implicating that the 54-kDa component was composed of component(s) of the same pI as that of the M protein monomers. From these results, we conclude that the M antigen-positive 54-kDa polypeptide is a homodimer of M protein, taking an N-terminal-exposed conformation, and is strongly associated with the viral glycoprotein. Possible association with a membrane microdomain of the cell will be discussed.     

The human leucocyte antigen CD48 (MEM-102) is closely related to the activation marker Blast-1

The glycosyl phosphatidylinositol (GPI)-linked antigen recognized by monoclonal antibody (mAb) MEM-102 is expressed on all peripheral blood lymphocytes, both resting and activated. Its properties are very similar to a previously described activation antigen, Blast-1. The amino acid sequence deduced from the structure of cloned cDNA is identical to that of the Blast-1 antigen except for a single amino acid residue. There are several other minor differences in the nucleotide sequence of the Blast-1 and MEM-102 cDNAs that do not affect the predicted structure of the polypeptide product. The amino acid sequence of the first 15 N-terminal residues of the antigen purified from Raji cells is found in the deduced sequence close to the presumed boundary between the leader peptide and mature polypeptide. Properties of the recombinant product expressed in COS cells are similar to the antigen isolated from peripheral blood mononuclear cells (PBMNCs) or B-and T-cells lines. The antigen purified on immobilized mAb MEM-102 is recognized by all six known CD48 mAbs under western blotting conditions. COS cells transfected with MEM-102 cDNA react with all the CD48 mAbs. It is concluded that mAb MEM-102 is directed against the as yet poorly characterized antigen CD48, which is therefore structurally closely related to Blast-1. Several possibilities are discussed that might account for the apparent discrepancy between the broad pan-leucocyte expression of the MEM-102/CD48 antigen and much more restricted expression of the epitope recognized by the previously described mAb defining the Blast-1 antigen.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 37766.     

Antigenic and Functional Analyses of Glycoprotein of Rabies Virus Using Monoclonal Antibodies

Thirty-five monoclonal antibodies (MAbs) against glycoprotein (G protein) of the RC-HL strain of the rabies virus have been established. Using these MAbs, two antigenic sites (I and II) were delineated on the G protein of the RC-HL strain in a competitive binding assay. Of these, 34 MAbs recognized the epitopes on site IL Site II was further categorized into 10 subsites according to their patterns in a competitive binding assay. Each site II-specific MAb showed 5 to 23 nonreciprocal competitions. The reactivities of 35 MAbs to rabies and rabies-related viruses in an indirect immunofluorescent antibody test showed that six MAbs in group A binded to rabies and rabies-related viruses and eight MAbs in group E reacted only with rabies viruses, considering that the former represent the genus-specific of Lyssavirus and the latter are rabies virus-specific. From biological assays, 28 of the 35 MAbs showed neutralization activity, 31 showed hemagglutination inhibition (HI) activity, and 18 showed immunolysis (IL) activity. The MAbs recognizing neutralization epitopes fell into at least three groups: those exhibiting both HI and IL activity, those showing only HI activity, and those showing neither HI nor IL activity. All IL epitopes overlap with HA epitopes. Five of the nine MAbs which reacted with the antigen treated by sodium dodecyl sulfate in ELISA were not reduced, or reduced only slightly, in the titer. None of the MAbs reacted with 2-mercaptoethanol-treated antigen. Only one MAb that recognized site I reacted with the denatured G protein in a Western blotting assay, indicating that its epitope is linear. These results suggest that almost all of the epitopes on the G protein of the rabies virus are conformation-dependent and the G protein forms a complicated antigenic structure.     

Intracellular behavior of rabies virus matrix protein (M) is determined by the viral glycoprotein (G).

To investigate the nature and intracellular behavior of the matrix (M) protein of an avirulent strain (HEP-Flury) of rabies virus, we cloned and sequenced the cDNA of the protein. Using expression vectors pZIP-NeoSV(X)1 and pCDM8, the cDNA was transfected to animal cells (BHK-21 and COS-7) with or without coexpression of viral glycoprotein (G). When M protein alone was expressed in the cells, it displayed homogeneous distribution in the whole cell including the nucleus. In contrast, coexpression with G protein resulted in the abolishment of nuclear distribution of M antigen, and both of the antigens displayed a colocalized distribution in the cell, especially at the cellular membrane as seen in the virus-infected cells, while the distribution of G antigen was not affected by coexpressed M antigen. Immunoprecipitation studies revealed that M protein was coprecipitated with G protein by anti-G antibody, and vice versa, although cross-linking with dithiobis(succinimidyl propionate) was necessary for coprecipitation because of their easier dissociation in the presence of sodium deoxycholate. These results suggest that M protein intimately associates with G protein, which may affect or regulate the behavior (e.g., intracellular localization) of M protein. Studies with deletion mutants of M protein indicate that an internal region around the amino acids from 115 to 151 is essential for the M protein to preserve its binding ability to G protein.     

Unprocessed foot-and-mouth disease virus capsid precursor displays discontinuous epitopes involved in viral neutralization.

A foot-and-mouth disease virus (FMDV) cDNA cassette containing sequences encoding the capsid precursor P1, peptide 2A and a truncated 2B (abbreviated P1-2A) of type C FMDV, has been modified to generate the authentic amino terminus and the myristoylation signal. This construct has been used to produce a recombinant baculovirus (AcMM53) which, upon infection of Spodoptera frugiperda insect cells, expressed a recombinant P1-2A precursor with a high yield. This polyprotein reacted with neutralizing monoclonal antibodies (MAbs) that bind to continuous epitopes of the major antigenic site A (also termed site 1) of capsid protein VP1. Unexpectedly, it also reacted with neutralizing MAbs which define complex, discontinuous epitopes previously identified on FMDV particles. The reactivity of MAbs with P1-2A was quantitatively similar to their reactivity with intact virus and, in both cases, the reactivity with MAbs that recognized discontinuous epitopes was lost upon heat denaturation of the antigen. The finding that a capsid precursor may fold in such a way as to maintain discontinuous epitopes involved in virus neutralization present on the virion surface opens the possibility of using unprocessed capsid precursors as novel antiviral immunogens.     

The polypeptide encoded by the cDNA for human cell surface antigen Fas can mediate apoptosis.

Mouse anti-Fas monoclonal antibody has a cytolytic activity on human cells that express the antigen. Complementary DNAs encoding the cell surface antigen Fas were isolated from a cDNA library of human T cell lymphoma KT-3 cells. The nucleotide sequence of the cDNAs revealed that the molecule coding for the Fas antigen determinant is a 319 amino acid polypeptide (Mr 36,000) with a single transmembrane domain. The extracellular domain is rich in cysteine residue, and shows a similarity to that of human tumor necrosis factor receptors, human nerve growth factor receptor, and human B cell antigen CD40. Murine WR19L cells or L929 cells transformed with the human Fas antigen cDNA were killed by the anti-Fas antibody in the process known as apoptosis.     

Immunochemical properties of West Nile virus protein prM and protein M C-end

Complementary DNA fragments (nucleotides 466-966 and 878-1088) encoding prM protein and polypeptide M31-75-E1-30 of West Nile virus (WNV), strain LEIV-Vlg99-27889-human, were obtained and cloned. Recombinant polypeptides prM and M3175-E1-30 having amino acid sequences corresponding to the cloned cDNA fragments were purified by affinity chromatography. According to ELISA and Western blotting prM protein interacted with polyclonal antibodies against WNV. This is indicative the immunochemical similarity of WNV recombinant and native protein prM. 6 types of species-specific monoclonal antibodies (MAbs) raised against recombinant polypeptide prM recognized at least four epitopes within recombinant polypeptides prM and M31-75-E1-30. MAbs 7D11 were active in the virus - neutralization assay. Analysis of interaction of the MAbs with recombinant polypeptides prM, M31-75-EI-30, E1-180, E260-466 revealed cross-reactive epitopes within 260-466 amino acid residues (aa) of WNV protein E, 31-75 aa of polypeptide M31-75-E1-30 and protein prM. Proposed spatial model of proteins E and M C-end fragments shown similarity of their three-dimensional structures confirming results of immunochemical assay. Neutralization of viral infectivity by MAbs 7D11 raised against epitope within 31-75 aa t of protein M is evidence of important function of C-end region in the process of flaviviral penetration into host cell.     

狂犬病病毒SRV_9突变株的构建与拯救

为构建人工修饰的狂犬病病毒,首先用人细胞色素C基因替换狂犬病病毒SRV9株基因间隔区中的非必需区域Ψ区并缺失基因组全长cDNA的糖蛋白CD编码区,得到突变型SRV9cDNA质粒。然后,该质粒与表达野生型SRV9四种结构蛋白N、P、G和L的质粒共转染BHK-21细胞。免疫荧光试验显示转染细胞中有大量特异性荧光,电子显微镜观察可见大量典型的狂犬病病毒粒子。上述结果表明已成功地拯救出了人工修饰的狂犬病病毒。狂犬病病毒SRV9突变株的成功构建与拯救,为新型狂犬病减毒活疫苗的研究提供了重要的实验工具。     

Studies on the regulation of 1-aminocyclopropane-1-carboxylate synthase in tomato using monoclonal antibodies

A partially purified preparation of 1-aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) from tomato (Lycopersicon esculentum (Mill.) fruit tissue was used to generate monoclonal antibodies (MAb) specific for the two different MAbs yielded a 50-kDa polypeptide as shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis. An enzyme-linked immunosorbent assay (ELISA) capable of detecting <1 ng of antigen was developed. The ELISA system was used to demonstrate that two of the MAbs recognized different epitopes on the ACC-synthase protein. Wound-induced increases in ACC-synthase activity in tomato fruit tissue were correlated with changes in ELISA-detectable protein. In-vivo labeling of wounded tissue with [³⁵S]methionine followed by extraction and immunopurification in the presence of various protease inhibitors yielded one major radioactive band of 50 kDa molecular mass. Pulse labeling with [³⁵S]methionine at various times after wounding indicated that the wound-induced increase in ACC-synthase activity involved de-novo synthesis of a rapidly turning over 50-kDa polypeptide.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - ELISA enzyme-linked immunosorbent assay - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Identification of porin-like polypeptide(s) in the boundary membrane of oilseed glyoxysomes

A 36-kDa polypeptide of unknown function was identified by us in the boundary membrane fraction of cucumber seedling glyoxysomes. Evidence is presented in this study that this 36-kDa polypeptide is a glyoxysomal membrane porin. A sequence of 24 amino acid residues derived from a CNBr-cleaved fragment of the 36-kDa polypeptide revealed 72% to 95% identities with sequences in mitochondrial or non-green plastid porins of several different plant species. Immunological evidence indicated that the 36-kDa (and possibly a 34-kDa polypeptide) was a porin(s). Antiserum raised against a potato tuber mitochondrial porin recognized on immunoblots 34-kDa and 36-kDa polypeptides in detergent-solubilized membrane fractions of cucumber seedling glyoxysomes and mitochondria, and in similar glyoxysomal fractions of cotton, castor bean, and sunflower seedlings. The 36-kDa polypeptide seems to be a constitutive component because it was detected also in membrane protein fractions derived from cucumber leaf-type peroxisomes. Compelling evidence that one or both of these polypeptides were authentic glyoxysomal membrane porins was obtained from electron microscopic immunogold analyses. Antiporin IgGs recognized antigen(s) in outer membranes of glyoxysomes and mitochondria. Taken together, the data indicate that membranes of cucumber (and other oilseed) glyoxysomes, leaf-type peroxisomes, and mitochondria possess similar molecular mass porin polypeptide(s) (34 and 36 kDa) with overlapping immunological and amino acid sequence similarities.