Characterization of the cell-cycle-regulated protein calcyclin from Ehrlich ascites tumor cells. Identification of two binding proteins obtained by Ca2(+)-dependent affinity chromatography

The nearly complete amino acid sequence obtained for murine calcyclin from Ehrlich ascites tumor cells reveals a very strong similarity with the rat and human sequences previously deduced from corresponding cDNA clones. While mouse and rat calcyclins are identical, the human protein shows at three positions a conservative amino acid replacement. Using a mouse calcyclin affinity matrix, two proteins with molecular masses of about 36 kDa have been purified from Ehrlich ascites tumor cells. The interaction between these two proteins and the immobilized calcyclin is strictly Ca2(+)-dependent. Immunological criteria and partial sequence data identify the two calcyclin-binding proteins as the phospholipid-binding protein annexin II (p36) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase. These observations suggest that calcyclin may exert its physiological function by a Ca2(+)-dependent interaction with cellular targets, e.g. annexin II or glyceraldehyde-3-phosphate dehydrogenase.     

Expression of Pichia anomala INV1 gene in Saccharomyces cerevisiae results in two different active forms of hypoglycosylated invertase

The Pichia anomala invertase gene (INV1) was introduced at different copy numbers into a sucrose-nonfermenting mutant of Saccharomyces cerevisiae and expressed from its own promoter sequences. The level reached in the production of invertase by the transformants (up to 540 units/10(10) cells) was in agreement with the INV1 gene dosage. Two forms of multimeric active and glycosylated invertase displaying different subcellular locations and molecular masses could be detected in the transformants. One was found to be present in the culture medium and in the periplasm, and the other could only be detected inside the cell. Each of the two heterologous forms of invertase was shown to be an oligomer composed of identical subunits. The difference found in the apparent molecular masses of their monomers (81.5 and 78.3 kDa, respectively) seems to be due to the size of their N-linked oligosaccharide chains (on average 2.4 and 1.9 kDa, respectively), since the number of sugar chains (9) and the molecular mass of the protein moiety (60.5 kDa) are identical in both forms. The shorter size of their oligosaccharides must also be the reason for the lower apparent molecular masses of the heterologous invertases when compared with the enzyme purified from P. anomala. The hypoglycosylated invertase accumulated within the cells of the transformants to an unusual level (up to 130 units/10(10) cells). Such accumulation of active enzyme inside the cells, as well as its underglycosylation, could be due to intrinsic properties of the P. anomala invertase that are determined by the particular primary structure of its protein moiety.     

Protein structural changes associated with active and inactive states of hydrogenase from Thiocapsa roseopersicina

Electrophoretic and isoelectrofocusing behavior of the hydrogenase from Thiocapsa roseopersicina under various conditions revealed remarkable properties of this enzyme: there are two active forms which differ in their molecular masses as well as in oxygen sensitivity; the apparent molecular masses of the active hydrogenase forms (90 and 49 kDa) differ considerably from those in the inactive state (64, 34, and 15 kDa); the active forms and some of the inactivated ones can be transformed into each other reversibly; urea can unfold the 64 and 34 kDa proteins but not the 49 kDa form at room temperature; the pI of these proteins are different in the presence of urea. The results suggest large rearrangements in the hydrogenase protein structure which are associated with the enzymatically active and inactive states. It is concluded that reversible formation of disulfide bonds cannot be the major cause for maintaining the enzyme conformation. Strong hydrophobic interactions are suggested to be primarily responsible for the structural stability and for the rearrangements.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

Changes in annexin (lipocortin) content in human amnion and chorion at parturition.

Arachidonic acid is mobilized from fetal membrane phospholipids at parturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometrial contractions. Phospholipid mobilization requires activation of phospholipase A2 or C, both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids and thereby may alter phospholipase activity through two mechanisms: modulation of intracellular free Ca2+ concentrations or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I-VI were identified in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pools: a pool which only associated with the membrane in the presence of calcium (calcium-dependent pool) and a calcium-independent pool that remained membrane bound in the presence of calcium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium-dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium-dependent pool of annexin V increased in chorion; and calcium-independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in total annexin I concentration with labor in amnion reflects a substantial decrease (80-90%) in the pool tightly bound to the membrane in a calcium-independent manner. This striking change distinguishes annexin I as a potential candidate inhibitor which is specifically downregulated at parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostaglandin production at labor.     

Biochemical Characterization of Annexins I and II Isolated from Pig Nervous Tissue

Five proteins having molecular masses of 90, 67, 37, 36, and 32 kDa (p90, p67, p37, p36, and p32, respectively) were identified in the particulate fractions of pig brain cortex and pig spinal cord prepared in the presence of 0.2 mM Ca2+ and further purified using a protocol previously described for the purification of calpactins. Proteins p90, p37, and p36 are related to annexins I and II. Annexin II, represented by p90, is found as an heterotetramer, composed of two heavy chains of 36 kDa and two light chains of 11 kDa, and as a monomer of 36 kDa. Protein p37, which differs immunologically from p36, is a monomer and could be related to annexin I. All three proteins are Ca(2+)-dependent phospholipid- and F-actin-binding proteins; they are phosphorylated on a serine and on a tyrosine residue by protein kinases associated with synaptic plasma membranes. Purified p36 monomer and p36 heterotetramer proteins bind to actin at millimolar Ca2+ concentrations. The stoichiometry of p36 binding to F-actin at saturation is 1:2, corresponding to one tetramer or monomer of calpactin for two actin monomers (KD, 3 x 10(-6) M). Synaptic plasma membranes supplemented with the monomeric or tetrameric forms of p36 phosphorylate the proteins on a serine residue. The monomer is phosphorylated on a serine residue by a Ca(2+)-independent protein kinase, whereas the heterotetramer is phosphorylated on a serine residue and a tyrosine residue by Ca(2+)-dependent protein kinases. Antibodies to brain p37 and p36 together with antibodies to lymphocytes lipocortins 1 and 2 were used to follow the distribution of these proteins in nervous tissues. Polypeptides of 37, 34, and 36 kDa cross-react with these antibodies. Anti-p37 and antilipocortin 1 cross-react on the same 37- and 34-kDa polypeptides; anti-p36 and antilipocortin 2 cross-react only on the 36-kDa polypeptides.     

Identification of epididymal proteins associated with hamster sperm.

The electrophoretic analysis of the proteins that were extracted from immature caput and mature cauda sperm showed evidence of accumulation of several proteins during the epididymal transit of the sperm. An antiserum, raised against detergent-extracted proteins from mature spermatozoa, immunostained six epididymal proteins with apparent molecular masses of 16, 22.5, 26, 37, 60, and 80 kDa on Western blots of epididymal fluid. Of these proteins, only the 26 kDa protein was significantly immunodetected in proximal caput epididymal fluid. Its biosynthesis by caput epididymis was confirmed by immunoprecipitation of an in vitro translated product of caput poly (A) RNA. The homology of the 26 kDa epididymal protein with the 26 kDa sperm protein was verified by epitope mapping. The other epididymal proteins were found in the fluid of the more distal portions of the organ. Their presence in the epididymal fluid coincided with their detection on the sperm. These epididymal proteins were considered to be sperm-coating proteins.     

Glycosylphosphatidylinositol is involved in the membrane attachment of proteins in granules of chromaffin cells.

Incubation at 37 degrees C or treatment of granule membranes of chromaffin cells with Staphylococcus aureus phosphatidylinositol-specific phospholipase C converted from an amphiphilic to a hydrophilic form two proteins with molecular masses of 82 and 68 kDa respectively. Their release is time- and enzyme-concentration-dependent. We showed that they were immunoreactive with an anti-(cross-reacting determinant) antibody known to be revealed only after removal of a diacylglycerol anchor. Furthermore, the action of HNO2 suggests the presence of a non-acetylated glucosamine residue in the determinant. This is one of the first reports suggesting that a glycosylphosphatidylinositol anchor might exist in membranes other than the plasma membrane. We showed that the 68 kDa protein is probably not the subunit of dopamine (3,4-dihydroxyphenethylamine) beta-hydroxylase, an enzyme present in granules in both soluble and membrane-associated forms.     

A series of annexins from human placenta and their characterization by use of an endogenous phospholipase A2.

Membranes from human placenta contain proteins which inhibit the activity of phospholipases A2 by binding to phospholipid thus impeding substrate availability. We used unilamellar mixed liposomes and a partially purified cytosolic phospholipase A2 from placenta for characterizing this substrate-depleting activity. A major portion of these inhibitory proteins was released by extracting washed membranes with a Ca+(+)-chelator. Biochemical fractionation and systematic analysis resulted in the unequivocal identification of a series of annexin proteins. We describe a straightforward procedure which allows to obtain 8 annexins from placenta either in pure form or as a mixture of two annexins. One of them was obtained in two forms which had the same molecular mass of 68 kDa but differed in charge. We also present suggestive evidence for a novel annexin I-related polypeptide of Mr 45,000 which is an excellent in vitro substrate for protein kinase C. We estimate that about 2% of the total placental membrane proteins are annexins. For achieving half inhibition of phospholipase A2 activity with pure annexins, up to a 6.5-fold difference in the amounts of protein was observed when calculated on a molar basis. This suggests specificity of individual annexin species.     

Glycosylation of procathepsin L does not account for species molecular-mass differences and is not required for proteolytic activity.

Cathepsin L is a major lysosomal cysteine proteinase in mouse and human cells. Despite similar predicted molecular masses, procathepsin L in these two species migrates on SDS/polyacrylamide gels with apparent molecular masses of 39 kDa and 42 kDa respectively. To determine if glycosylation differences account for this discrepancy, and to ascertain whether glycosylation is essential for enzymic activity, mouse and human procathepsins L were expressed at high concentrations in mouse NIH 3T3 cells or in human A431 cells after DNA-mediated transfection of cloned DNAs for these enzymes. In pulse-chase studies, human procathepsin L transfectants synthesized and secreted large amounts of enzymically active 42 kDa proenzyme and processed it into 34 kDa and 26 kDa intracellular peptides, a pattern of secretion and processing similar to that seen with endogenous or transfected mouse procathepsin L. Both translation of cloned procathepsin L cDNAs in vitro and Endoglycosidase H treatment of 39 kDa mouse and 42 kDa human procathepsin L resulted in non-glycosylated proteins 2 kDa lower in molecular mass than the untreated proteins for both species. This suggests that glycosylation differences are not responsible for the molecular-mass disparity between the two species. Moreover, Endoglycosidase H-treated mouse enzyme retained full proteolytic activity, indicating that glycosylation of cathepsin L is not essential for enzymic function.     

Characterization by Hg2+ of two different pathways for mitochondrial Ca2+ release

The addition of Hg2+ to loaded kidney mitochondria induces the fast release of the accumulated cation. The Ca2+-efflux reaction exhibits kinetics characteristics that depend on the extent of the binding of Hg2+ to the membrane. At high levels of Hg2+ bound (approx. 11 nmol/mg), Ca2+ efflux rate is highly insensitive to the temperature of incubation, and the efflux seems to be directly related to the internal free Ca2+ concentration. At these levels of bound Hg2+, accumulated Sr2+ is released with characteristics similar to those observed with Ca2+. At lower levels of Hg2+ binding (2.5 nmol/mg), the efflux reaction is highly dependent on the incubation temperature and on the internal free Ca2+ concentration; under these conditions Sr2+ is not released. NAD(P)H oxidation as induced by the low Hg2+ concentration is inhibited at the lower temperatures. Radiolabeled Hg2+ incorporates into two clearly defined regions of membrane proteins separated through sodium dodecyl sulfate gel electrophoresis. One of the regions corresponds to proteins of apparent high molecular mass (i.e., 150 kDa), and the other to proteins with apparent molecular masses of 37-25 kDa. Mitochondria incubated with 2 microM 203Hg2+ incorporate the radionuclide in proteins that have molecular masses of around 41 and 26 kDa. The results indicate that, depending on the amount of Hg2+ bound to the inner membrane, two clearly distinct Ca2+ release mechanisms can be distinguished.     

Phosphorylation of proteins in human neutrophils activated with phorbol myristate acetate or with chemotactic factor

In human neutrophils stimulated with phorbol myristate acetate (PMA) or with the chemotactic factor N-formyl-methionyl-leucyl-phenylalanine (fMLF) a number of proteins are phosphorylated, including proteins recovered in the membrane fraction corresponding to molecular masses of 130, 78, 46, 40, and 34 kDa and proteins recovered in the cytosol fraction corresponding to molecular masses of 65, 55, 48, 38, 36, 30, and 22 kDa. Phosphorylation of the membrane proteins was fourfold greater in cells stimulated with PMA, as compared to cells stimulated with fMLF, whereas both activators induced similar phosphorylation of proteins recovered in the cytosol fraction. Phosphorylation of membrane proteins appeared to be mediated by native protein kinase C (PKC) translocated from the cytosol to the plasma membrane. Thus phosphate incorporation was inhibited by retinal and a similar pattern of incorporation was reproduced in a reconstituted system composed of isolated cell membranes and purified PKC. Phosphorylation of cytosol proteins, on the other hand, appeared to be mediated by the proteolytically modified form of PKC. In this case, phosphate incorporation was inhibited by leupeptin, which prevents the conversion of native PKC to the proteolytically modified form, The phosphorylation pattern was reproduced when isolated cytosol fractions were incubated with the proteolytically modified form of the enzyme but not with the native PKC. These results demonstrate that responses to stimuli such as PMA or fMLF are mediated by different forms of PKC and that the proteolytically modified form is responsible for the major responses elicited by fMLF.     

Identification of phosphoproteins associated with maintenance of transformed state in temperature-sensitive Rous sarcoma-virus infected cells by proteomic analysis

To identify phosphotyrosine-containing proteins essential for maintaining the transformed state, we studied the tyrosine phosphorylation profile of temperature-sensitive mutant of Rous sarcoma virus, tsNY68, infected cells (68N7). Shifting the temperature from 39 degrees C (nonpermissive) to 32 degrees C (permissive) markedly increased the expression of phosphotyrosine-containing cell membrane proteins of approximately 40kDa, as assessed by SDS-PAGE. Membrane and nuclear proteins were separated by two-dimensional gel electrophoresis and immunoblotted with anti-phosphotyrosine antibody. Proteins showing temperature-dependent changes in phosphorylation profile were subjected to in-gel digestion with trypsin and analyzed by mass spectrometry. Five proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A3, hnRNP A2, annexin II, phosphoglycerate mutase 1, and triosephosphate isomerase 1. hnRNP A3 was phosphorylated at serine residues and had both serine and tyrosine phosphorylated sites. These results suggest an important complementary role for proteomics in identifying molecular abnormalities associated with tumor progression that may be attractive candidates for tumor diagnosis.     

Outer membrane proteins of Fusobacterium nucleatum Fev1

Outer membrane enriched material from six strains of Fusobacterium nucleatum was analysed by SDS-PAGE. The protein profiles of all the strains were dominated by proteins with molecular masses of about 40 kDa, and a very high degree of homology in relation to apparent molecular masses was observed. In all strains except Fev1, one of the most dominant proteins exhibited heat modifiable properties, having an apparent molecular mass of about 38 kDa and 42 kDa when heated in SDS at 50 and 100 degrees C, respectively. None of the proteins of the outer membrane of F. nucleatum Fev1 demonstrated such heat modifiable properties. The 40 kDa protein, and several other proteins, appear to be both exposed on the cell surface and peptidoglycan associated.     

SH3 binding sites of ZG29p mediate an interaction with amylase and are involved in condensation-sorting in the exocrine rat pancreas

ZG29p, a novel pancreas-specific zymogen granule protein, has been proposed to act as a 'helper protein' in granule formation. To address its function in more detail, we searched for putative binding partners of ZG29p. In zymogen complexes isolated by nondenaturing isoelectric focusing, ZG29p was associated with a protein complex consisting of amylase and cationic trysinogen. Amylase also coeluted with ZG29p after immunoaffinity chromatography using an antibody to recombinant ZG29p. Cross-linking experiments with granule content proteins revealed a direct interaction between recombinant ZG29p and amylase. An interaction was also observed when purified amylase was used, whereas no interaction with recombinant or purified cationic trypsinogen was seen. ZG29p could also be cross-linked to three membrane proteins with molecular masses of 40, 18, and 16 kDa. The binding of ZG29p to amylase and to the membrane proteins was inhibited in the presence of synthetic peptides matching the consensus sequence of proline-rich SH3 binding sites present in ZG29p. The synthetic peptides could be cross-linked to amylase and to three yet unidentified acidic content proteins with molecular masses of about 30 kDa. The peptides also interacted with purified or recombinant amylase, but not with recombinant or purified cationic trypsinogen. In a condensation-sorting assay, the binding (sorting) of zymogen complexes to the granule membrane was reduced in the presence of the peptides. Our results indicate that the interaction of ZG29p with amylase is mediated by SH3 binding domains and that these domains are involved in the sorting of amylase to the granule membrane.     

Purification and characterization of S layer proteins from Clostridium difficile GAI 0714

The S layer of Clostridium difficile GAI0714 was shown to be composed of two proteins, of 32 kDa and 45 kDa, as determined by SDS-PAGE. The two proteins were extracted with 8 M-urea (pH 8.3) from a cell wall preparation and purified by DEAE-Sepharose CL-6B chromatography followed by HPLC gel filtration. When solubilized in 0.1 M-urea, both proteins appeared to exhibit dimeric forms, with respective molecular masses of about 61 kDa and 99 kDa, upon HPLC. Although the amino acid compositions of the two proteins differed from each other, both proteins had a high content of acidic amino acids, very low contents of histidine and methionine, and no cysteine. The 32 kDa protein exhibited multiple isoelectric forms (pI 3.7-3.9), whereas the 45 kDa protein had a single form (pI 3.3). Radioiodination and immunogold labelling revealed that both proteins were exposed evenly over the entire cell surface. Based on immunodiffusion analysis using monospecific antiserum raised to the individual proteins, there was no antigenic relationship between the two proteins. Furthermore, immunoblot analysis showed that the antigenicity of the 32 kDa protein appeared to be strain specific, whereas that of the 45 kDa protein appeared to be group specific.     

Spectroscopic and molecular characterization of the oligomeric antenna of the diatom Phaeodactylum tricornutum

The photosynthetic antenna system of diatoms contains fucoxanthin chlorophyll a/c binding proteins (FCPs), which are membrane intrinsic proteins showing high homology to the light harvesting complexes (LHC) of higher plants. In the present study, we used a mild solubilization of P. tricornutum thylakoid membranes in combination with sucrose density gradient centrifugation or gelfiltration and obtained an oligomeric FCP complex (FCPo). The spectroscopic characteristics and pigment stoichiometries of the FCPo complex were comparable to FCP complexes that were isolated after solubilization with higher detergent per chlorophyll ratios. The excitation energy transfer between the FCP-bound pigments was more efficient in the oligomeric FCPo complexes, indicating that these complexes may represent the native form of the diatom antenna system in the thylakoid membrane. Determination of the molecular masses of the two different FCP fractions by gelfiltration revealed that the FCP complexes consisted of trimers, whereas the FCPo complexes were either composed of six monomers or two tightly associated trimers. In contrast to vascular plants, stable functional monomers could not be isolated in P. tricornutum. Both types of FCP complexes showed two protein bands in SDS-gels with apparent molecular masses of 18 and 19 kDa, respectively. Sequence analysis by MS/MS revealed that the 19 kDa protein corresponded to the fcpC and fcpD genes, whereas the 18 kDa band contained the protein of the fcpE gene. The presence of an oligomeric antenna in diatoms is in line with the oligomeric organization of antenna complexes in different photoautotrophic groups.     

Microtubule-associated proteins bind to 30 kDa and 60 kDa proteins of rat brain mitochondria: visualization by ligand blotting

Axonal transport of mitochondria is a microtubule-associated movement. Microtubule-mitochondria interactions were studied in vitro using organelles isolated from rat brain. Thanks to the ligand blotting method we were able to show two mitochondrial membrane proteins with apparent molecular masses of 30 kDa and 60 kDa that bind microtubule-associated proteins. The binding of the 30 kDa protein has an apparent Kd of 8 x 10(-8) M. Digitonin fractionation of mitochondria reveals a bimodal localization of the 30 kDa and the 60 kDa proteins within the outer membrane. The data suggest that these polypeptides could participate to the interactions observed in situ between microtubules and mitochondria.     

The Fusarium solani-induced expression of a pea gene family encoding high cysteine content proteins.

Two pea genes, pI39 and pI230, which are specifically induced by two forma specials of Fusarium solani, encode closely related proteins with predicted molecular masses (Mr) of 8.2 and 8 kDa, respectively. Both proteins contain a signal sequence and are cleaved to mature proteins of Mr 5 kDa as indicated by an in vitro translation system. The mature proteins contain about 17% cysteine residues and have the potential to form four disulfide bonds. The two proteins share extensive homology in their signal sequences but much less homology as mature proteins. The cysteine residues of the mature proteins are highly conserved, suggesting functional importance. Southern hybridization suggests these genes belong to a multigene family. The relative accumulations of mRNA levels indicate that the two genes are expressed somewhat differentially. In both the compatible (susceptible) and incompatible reactions between F. solani and pea tissue, pI39 mRNA accumulates more slowly than pI230 mRNA and accumulates to relatively high levels after 24 hr of inoculation. The increase in accumulation of pI230 mRNA occurs within 6 hr and thus correlates with an initial suppression of the growth of both the compatible and incompatible pathogen, which is cytologically observable at 6 hr. pI39 and pI230 belong to a distinct class of pathogenesis-related proteins characterized previously, which are associated with and thus may contribute to nonhost resistance in plants.     

Hepatitis B virus p25 precore protein accumulates in Xenopus oocytes as an untranslocated phosphoprotein with an uncleaved signal peptide.

We have analyzed the translocation of hepatitis B virus (HBV) precore (PC) proteins by using Xenopus oocytes injected with a synthetic PC mRNA. The PC region is a 29-amino-acid sequence that precedes the 21.5-kDa HBV capsid or core (C) protein (p21.5) and directs the secretion of core-related proteins. The first 19 PC amino acids provide a signal peptide that is cleaved with the resultant translocation of a 22.5-kDa species (p22.5), in which the last 10 PC residues precede the complete p21.5 C polypeptide. Most p22.5 is matured to 16-20 kDa species by carboxyl-terminal proteolytic cleavage prior to secretion. Here we show that some four unexpected PC proteins of 24 to 25 kDa are produced in addition to the secretion products described above. Protease protection and membrane cosedimentation experiments reveal that all PC proteins behave as expected for proteins that are translocated into the lumen of the endoplasmic reticulum except for the single largest PC protein (p25), which is not translocated. Like p21.5, p25 is a phosphoprotein that localizes to the oocyte cytosol and nucleus, and protease digestion studies suggest that the two molecules have similar two-domain structures. Radiosequencing of immobilized p25 demonstrates that it contains the intact PC signal peptide and represents the unprocessed translation product of the entire PC/C locus. Thus, while many HBV PC protein molecules are correctly targeted to intracellular membranes and translocated, a significant fraction of these molecules can evade translocation and processing.