Organization of translocon complexes in ER membranes.

Protein translocation in the ER (endoplasmic reticulum) and N-glycosylation are fundamental processes essential for the normal functioning of eukaryotic cells. They are the initial steps in the intracellular pathway that are followed by secretory proteins and membrane proteins of the endomembrane system and the plasma membrane. The translocation and concurrent N-glycosylation of these proteins take place on a large molecular machine, the TC (translocon complex), which is associated with membrane-bound polysomes. Segregation of TCs into a differentiated domain of the ER, the rough ER, may increase the efficiency of protein synthesis on membrane-bound polysomes. Our research is concerned with the assembly, functional organization and dynamics of the TCs in the ER, and their contribution to the functioning and the morphological appearance of this organelle. We hypothesize that the TCs form higher-order structures defining the rough domain of the ER. These structures, which are immobilized or diffuse slowly in the plain of the ER membrane, may be formed and stabilized by mRNAs interconnecting the TCs, by cytoskeletal elements and/or by hypothetical proteins that form links between the TCs. We have established the M3/18 cell line, which expresses the GFP (green fluorescent protein)-Dad1 fusion protein quantitatively and functionally incorporated into the OST (oligosaccharyltransferase). GFP-Dad1 can be used as a reporter molecule for the lateral mobility of the TCs since the OST is tightly associated with the complex. As determined by FRAP (fluorescence recovery after photobleaching), the lateral mobility of GFP-Dad1-tagged TCs was much more restricted than expected from the estimated size of the TC and can be affected by the functional state of the TCs. Currently, we are studying the possible involvement of cytoskeletal elements in the organization of the TCs. Our data suggest that microtubules also play a role in the immobilization of the TCs.

Cholesterol and steroid synthesizing smooth endoplasmic reticulum of adrenocortical cells contains high levels of translocation apparatus proteins.

Steroid-secreting cells possess abundant smooth endoplasmic reticulum whose membranes contain many enzymes involved in sterol and steroid synthesis. In this study we demonstrate that adrenal smooth microsomal subfractions enriched in these membranes also possess high levels of proteins belonging to the translocation apparatus, proteins previously assumed to be confined to morphologically identifiable rough endoplasmic reticulum (RER). We further demonstrate that these smooth microsomal subfractions are capable of effecting the functions of these protein complexes: co-translational translocation, signal peptide cleavage and N-glycosylation of newly synthesized polypeptides. We hypothesize that these elements participate in regulating the levels of ER-targeted membrane proteins involved in cholesterol and steroid metabolism in a sterol-dependent and hormonally-regulated manner.

In vitro binding of ribosomes to the beta subunit of the Sec61p protein translocation complex.

The Sec61p complex forms the core element of the protein translocation complex (translocon) in the rough endoplasmic reticulum (rough ER) membrane. Translating or nontranslating ribosomes bind with high affinity to ER membranes that have been stripped of ribosomes or to liposomes containing purified Sec61p. Here we present evidence that the beta subunit of the complex (Sec61beta) makes contact with nontranslating ribosomes. A fusion protein containing the Sec61beta cytoplasmic domain (Sec61beta(c)) prevents the binding of ribosomes to stripped ER-derived membranes and also binds to ribosomes directly with an affinity close to the affinity of ribosomes for stripped ER-derived membranes. The ribosome binding activity of Sec61beta(c), like that of native ER membranes, is sensitive to high salt concentrations and is not based on an unspecific charge-dependent interaction of the relatively basic Sec61beta(c) domain with ribosomal RNA. Like stripped ER membranes, the Sec61beta(c) sequence binds to large ribosomal subunits in preference over small subunits. Previous studies have shown that Sec61beta is inessential for ribosome binding and protein translocation, but translocation is impaired by the absence of Sec61beta, and it has been proposed that Sec61beta assists in the insertion of nascent proteins into the translocation pore. Our results suggest a physical interaction of the ribosome itself with Sec61beta; this may normally occur alongside interactions between the ribosome and other elements of Sec61p, or it may represent one stage in a temporal sequence of binding.

Localization of ribophorin II to the endoplasmic reticulum involves both its transmembrane and cytoplasmic domains.

Proteins that are concentrated in specific compartments of the endomembrane system in order to exert their organelle-specific function must possess specific localization signals that prevent their transport to distal regions of the exocytic pathway. Some resident proteins of the endoplasmic reticulum (ER) that are known to escape with low efficiency from this organelle to a post ER compartment are recognized by a recycling receptor and brought back to their site of residence. Other ER proteins, however, appear to be retained in the ER by mechanisms that operate in the organelle itself. The mammalian oligosaccharyltransferase (OST) is a protein complex that effects the cotranslational N-glycosylation of newly synthesized polypeptides, and is composed of at least four rough ER-specific membrane proteins: ribophorins I and II (RI and RII), OST48, and Dadl. The mechanism(s) by which the subunits of this complex are retained in the ER are not well understood. In an effort to identify the domains within RII responsible for its ER localization we have studied the fate of chimeric proteins in which one or more RII domains were replaced by the corresponding ones of the Tac antigen, the latter being a well characterized plasma membrane protein that lacks intrinsic ER retention signals and serves to provide a neutral framework for the identification of retention signals in other proteins. We found that the luminal domain of RII by itself does not contain retention information, while the cytoplasmic and transmembrane domains contain independent ER localization signals. We also show that the retention function of the transmembrane domain is strengthened by the presence of a flanking luminal region consisting of 15 amino acids.

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum.

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.

Degradation of a short-lived glycoprotein from the lumen of the endoplasmic reticulum: the role of N-linked glycans and the unfolded protein response.

We are studying endoplasmic reticulum-associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI(332), containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI(332) was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of approximately 45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI(332) in which the single used N-glycosylation consensus site had been removed (RI(332)-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI(332) degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI(332) to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI(332). After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI(332) and RI(332)-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI(332) and RI(332)-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI(332) was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

A general mechanism for regulation of access to the translocon: competition for a membrane attachment site on ribosomes.

For proteins to enter the secretory pathway, the membrane attachment site (M-site) on ribosomes must bind cotranslationally to the Sec61 complex present in the endoplasmic reticulum membrane. The signal recognition particle (SRP) and its receptor (SR) are required for targeting, and the nascent polypeptide associated complex (NAC) prevents inappropriate targeting of nonsecretory nascent chains. In the absence of NAC, any ribosome, regardless of the polypeptide being synthesized, binds to the endoplasmic reticulum membrane, and even nonsecretory proteins are translocated across the endoplasmic reticulum membrane. By occupying the M-site, NAC prevents all ribosome binding unless a signal peptide and SRP are present. The mechanism by which SRP overcomes the NAC block is unknown. We show that signal peptide-bound SRP occupies the M-site and therefore keeps it free of NAC. To expose the M-site and permit ribosome binding, SR can pull SRP away from the M-site without prior release of SRP from the signal peptide.

DAD1 is required for the function and the structural integrity of the oligosaccharyltransferase complex.

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.

The bladder as a bioreactor: urothelium production and secretion of growth hormone into urine.

Uroplakin genes are expressed in a bladder-specific and differentiation-dependent fashion. Using a 3.6-kb promoter of mouse uroplakin II gene, we have generated transgenic mice that express human growth hormone (hGH) in their bladder epithelium, resulting in its secretion into the urine at 100-500 ng/ml. The levels of urine hGH concentration remain constant for longer than 8 months. hGH is present as aggregates mostly in the uroplakin-delivering cytoplasmic vesicles that are targeted to fuse with the apical surface. Using the bladder as a bioreactor offers unique advantages, including the utility of all animals throughout their lives. Using urine, which contains little protein and lipid, as a starting material facilitates recombinant protein purification.

Unregulated exposure of the ribosomal M-site caused by NAC depletion results in delivery of non-secretory polypeptides to the Sec61 complex.

Nascent polypeptide associated complex (NAC) interacts with nascent polypeptides emerging from ribosomes. Both signal recognition particle (SRP) and NAC work together to ensure specificity in co-translational targeting by competing for binding to the ribosomal membrane attachment site. While SRP selects signal-containing ribosomes for targeting, NAC prevents targeting of signal peptide-less nascent chains to the endoplasmic reticulum membrane. Here we show that the ribosome binding that occurs in NAC's absence delivers signalless nascent chains to the Sec61 complex, underscoring the danger of unregulated exposure of the ribosomal M-site. Recently, the idea that NAC prevents ribosome binding has been challenged. By carefully examining the physiologic NAC concentration in a variety of tissues from different species we here demonstrate that the discrepancy resulted from subphysiologic NAC concentrations.

Interactions among subunits of the oligosaccharyltransferase complex.

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Functional protein prenylation is required for the brefeldin A-dependent retrograde transport from the Golgi apparatus to the endoplasmic reticulum.

In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells. Upon treatment of the cells with BFA, RI332 becomes quantitatively O-glycosylated by Golgi glycosyltransferases that are transported back to the ER. Here we demonstrate that pretreatment of the cells with lovastatin, an inhibitor of HMG-CoA reductase, abrogates this modification and that mevalonate, the product formed in the step inhibited by the drug, is able to counteract the effect of lovastatin. We also show by immunofluorescence using mannosidase II as a Golgi marker that the BFA-induced retrograde transport of Golgi enzymes is blocked by lovastatin, although electron microscopy indicates that BFA causes disassembly of the Golgi apparatus into swollen vesicles and tubules. Our observations support the role of a prenylated protein, such as the geranylgeranylated small G protein Rab6, in the retrograde transport from the Golgi apparatus to the ER, since lovastatin acts by inhibiting its prenylation.

Incomplete endoplasmic reticulum (ER) retention in immature thymocytes as revealed by surface expression of "ER-resident" molecular chaperones.

The folding and assembly of nascent proteins in the endoplasmic reticulum (ER) is assisted by molecular chaperones that are themselves retained within the ER. We now report that a number of different ER proteins, including molecular chaperones, are selectively expressed on the surface of immature thymocytes, but their surface expression is extinguished upon further differentiation. Escape from the ER is only possible for newly synthesized ER proteins before they become permanently retained. Thus, the cellular process of ER retention is incomplete in immature thymocytes and provides an explanation for surface expression of partial receptor complexes that transduce differentiative signals during thymic development.

Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153).

The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damage-inducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2'-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression.

The intrinsic ability of ribosomes to bind to endoplasmic reticulum membranes is regulated by signal recognition particle and nascent-polypeptide-associated complex.

Signal peptides direct the cotranslational targeting of nascent polypeptides to the endoplasmic reticulum (ER). It is currently believed that the signal recognition particle (SRP) mediates this targeting by first binding to signal peptides and then by directing the ribosome/nascent chain/SRP complex to the SRP receptor at the ER. We show that ribosomes can mediate targeting by directly binding to translocation sites. When purified away from cytosolic factors, including SRP and nascent-polypeptide-associated complex (NAC), in vitro assembled translation intermediates representing ribosome/nascent-chain complexes efficiently bound to microsomal membranes, and their nascent polypeptides could subsequently be efficiently translocated. Because removal of cytosolic factors from the ribosome/nascent-chain complexes also resulted in mistargeting of signalless nascent polypeptides, we previously investigated whether readdition of cytosolic factors, such as NAC and SRP, could restore fidelity to targeting. Without SRP, NAC prevented all nascent-chain-containing ribosomes from binding to the ER membrane. Furthermore, SRP prevented NAC from blocking ribosome-membrane association only when the nascent polypeptide contained a signal. Thus, NAC is a global ribosome-binding prevention factor regulated in activity by signal-peptide-directed SRP binding. A model presents ribosomes as the targeting vectors for delivering nascent polypeptides to translocation sites. In conjunction with signal peptides, SRP and NAC contribute to this specificity of ribosomal function by regulating exposure of a ribosomal membrane attachment site that binds to receptors in the ER membrane.

The Brefeldin A-induced retrograde transport from the Golgi apparatus to the endoplasmic reticulum depends on calcium sequestered to intracellular stores.

Ribophorin I is a type I transmembrane glycoprotein specific to the rough endoplasmic reticulum. We have previously shown that, when expressed in transfected HeLa cells, a carboxyl-terminally truncated form of ribophorin I that contains most of the luminal domain (RI332) is, like the native protein, retained in the endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa cells leads to O-glycosylation of RI332 by glycosyltransferases that are redistributed from the Golgi apparatus to the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S., Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of glycosylation of RI332 as a measure for the BFA-induced backflow of enzymes of the Golgi apparatus to the ER, we now demonstrate that the retrograde transport is inhibited when cells are treated with various agents that affect intracellular Ca2+ concentrations, such as the dipeptide benzyloxycarbonyl (Cbz)-Gly-Phe-amide, the Ca2+ ionophore A23187, and thapsigargin, an inhibitor of the Ca(2+)-transporting ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI332. Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca2+ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of beta-COP from the Golgi apparatus. We conclude that the maintenance of a Ca2+ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFA-induced retrograde transport from the Golgi apparatus to the ER to occur.

Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

Functional characterization of the cis-regulatory elements of the rat ribophorin I gene.

The ribophorin I gene encodes a rough endoplasmic reticulum (RER) specific membrane protein which is a subunit of the oligosaccharyltransferase. To establish the functional activity of its promoter region we have performed transient gene transcription experiments employing plasmid constructs that contain 5' flanking regions of the ribophorin I gene cloned upstream of the CAT reporter gene. Among the restriction fragments obtained from the 1.3-kilobase 5' flanking region, a proximal fragment (-42 to +24) containing two GC-rich elements was required for basic promoter activity, while a fragment (-364 to +24) encoding an additional GC-box and an octamer like motif at -233 conferred the maximal promoter activity. In order to investigate the functionality of an octomer-like sequence co-transfection experiments were performed with Oct-2 cDNA and the CAT reporter gene containing the ribophorin I fragment (-364 to +24). A 3-4-fold increase in the transcriptional activity was observed with this construct. In addition, gel shift experiments showed Oct-2 binding to this construct. These results indicate that Oct-2 is most likely involved in the regulation of the ribophorin I gene transcription. We suggest that the GC-rich elements are necessary for constitutive ribophorin I expression while octamer motif binding proteins function synergistically with the GC-rich element binding proteins to increase the expression of the ribophorin I gene during the proliferation of RER.

Precursor sequence, processing, and urothelium-specific expression of a major 15-kDa protein subunit of asymmetric unit membrane.

The asymmetric unit membrane (AUM) is a highly specialized biomembrane elaborated by terminally differentiated urothelial cells. It contains quasi-crystalline arrays of 12-nm protein particles each of which is composed of six dumbbell-shaped subdomains. In this paper we describe the precursor sequence, processing and in vitro membrane insertion properties of bovine uroplakin II (UPII), a 15-kDa major protein component of AUM. The cDNA-deduced amino acid sequence revealed that UPII is synthesized as a precursor protein containing a cleavable signal peptide of approximately 26 amino acids, a long pro-sequence of approximately 59 residues harboring three potential N-glycosylation sites, and the mature polypeptide of 100 residues. In vitro translation of UPII mRNA demonstrated that UPII is indeed first synthesized as a 19-kDa precursor, which loses its signal peptide upon insertion into added microsomes; this process is accompanied by the acquisition of high mannose-type oligosaccharides giving rise to a 28-kDa precursor which is completely protected from the digestion by exogenous proteases. These results, together with the presence of a stretch of 25 hydrophobic amino acids at the C terminus, suggest that UPII protein is anchored to the lipid bilayer via its C-terminal membrane-spanning domain with its major N-terminal domain exposed luminally. The formation of the 15-kDa mature UPII requires the removal of the pro-sequence by a furin-like endoprotease. Since only mature UPII devoid of this pro-sequence can interact with 27-kDa uroplakin I, the proteolytic processing of UPII precursor may play an important role in regulating the assembly of AUM. Finally, we showed that genomic sequences cross-hybridizing with bovine UPII cDNA are present in many mammals suggesting that UPII performs a highly conserved function in the terminally differentiated cells of mammalian urinary bladder epithelium.