Transgene expression of prion protein induces crinophagy in intermediate pituitary cells

The cellular form of the prion protein (PrP(C)) is a plasma membrane-anchored glycoprotein whose physiological function is poorly understood. Here we report the effect of transgene expression of Xenopus PrP(C) fused to the C-terminus of the green fluorescent protein (GFP-PrP(C)) specifically in the neuroendocrine intermediate pituitary melanotrope cells of Xenopus laevis. In the transgenic melanotrope cells, the level of the prohormone proopiomelanocortin (POMC) in the secretory pathway was reduced when the cells were (i) exposed for a relatively long time to the transgene product (by physiologically inducing transgene expression), (ii) metabolically stressed, or (iii) forced to produce unfolded POMC. Intriguingly, although the overall ultrastructure was normal, electron microscopy revealed the induction of lysosomes taking up POMC secretory granules (crinophagy) in the transgenic melanotrope cells, likely causing the reduced POMC levels. Together, our results indicate that in neuroendocrine cells transgene expression of PrP(C) affects the functioning of the secretory pathway and induces crinophagy.     

Cell type-specific transgene expression of the prion protein in Xenopus intermediate pituitary cells

The cellular form of prion protein (PrPC) is anchored to the plasma membrane of the cell and expressed in most tissues, but predominantly in the brain, including in the pituitary gland. Thus far, the biosynthesis of PrPC has been studied only in cultured (transfected) tumour cell lines and not in primary cells. Here, we investigated the intracellular fate of PrPCin vivo by using the neuroendocrine intermediate pituitary melanotrope cells of the South-African claw-toed frog Xenopus laevis as a model system. These cells are involved in background adaptation of the animal and produce high levels of its major secretory cargo proopiomelanocortin (POMC) when the animal is black-adapted. The technique of stable Xenopus transgenesis in combination with the POMC gene promoter was used as a tool to express Xenopus PrPC amino-terminally tagged with the green fluorescent protein (GFP-PrPC) specifically in the melanotrope cells. The GFP-PrPC fusion protein was expressed from stage-25 tadpoles onwards to juvenile frogs, the expression was induced on a black background and the fusion protein was subcellularly located mainly in the Golgi apparatus and at the plasma membrane. Pulse-chase metabolic cell labelling studies revealed that GFP-PrPC was initially synthesized as a 45-kDa protein that was subsequently stepwise glycosylated to 48-, 51-, and eventually 55-kDa forms. Furthermore, we revealed that the mature 55-kDa GFP-PrPC protein was sulfated, anchored to the plasma membrane and cleaved to a 33-kDa product. Despite the high levels of transgene expression, the subcellular structures as well as POMC synthesis and processing, and the secretion of POMC-derived products remained unaffected in the transgenic melanotrope cells. Hence, we studied PrPC in a neuroendocrine cell and in a well-defined physiological context.     

Endocytic intermediates involved with the intracellular trafficking of a fluorescent cellular prion protein

We have investigated the intracellular traffic of PrP(c), a glycosylphosphatidylinositol (GPI)-anchored protein implicated in spongiform encephalopathies. A fluorescent functional green fluorescent protein (GFP)-tagged version of PrP(c) is found at the cell surface and in intracellular compartments in SN56 cells. Confocal microscopy and organelle-specific markers suggest that the protein is found in both the Golgi and the recycling endosomal compartment. Perturbation of endocytosis with a dynamin I-K44A dominant-negative mutant altered the steady-state distribution of the GFP-PrP(c), leading to the accumulation of fluorescence in unfissioned endocytic intermediates. These pre-endocytic intermediates did not seem to accumulate GFP-GPI, a minimum GPI-anchored protein, suggesting that PrP(c) trafficking does not depend solely on the GPI anchor. We found that internalized GFP-PrP(c) accumulates in Rab5-positive endosomes and that a Rab5 mutant alters the steady-state distribution of GFP-PrP(c) but not that of GFP-GPI between the plasma membrane and early endosomes. Therefore, we conclude that PrP(c) internalizes via a dynamin-dependent endocytic pathway and that the protein is targeted to the recycling endosomal compartment via Rab5-positive early endosomes. These observations indicate that traffic of GFP-PrP(c) is not determined predominantly by the GPI anchor and that, different from other GPI-anchored proteins, PrP(c) is delivered to classic endosomes after internalization.     

Characterization of the properties and trafficking of an anchorless form of the prion protein

Conversion of PrP(C) into PrP(Sc) is the central event in the pathogenesis of transmissible prion diseases. Although the molecular basis of this event and the intracellular compartment where it occurs are not yet understood, the association of PrP with cellular membranes and in particular its presence in detergent-resistant microdomains appears to be of critical importance. In addition it appears that scrapie conversion requires membrane-bound glycosylphosphatidylinositol (GPI)-linked PrP. The GPI anchor may affect either the conformation, the intracellular localization, or the association of the prion protein with specific membrane domains. However, how this occurs is not known. To understand the relevance of the GPI anchor for the cellular behavior of PrP, we have studied the biosynthesis and localization of a PrP version which lacks the GPI anchor attachment signal (PrP Delta GPI). We found that PrP Delta GPI is tethered to cell membranes and associates to membrane detergent-resistant microdomains but does not assume a transmembrane topology. Differently to PrP(C), this protein does not localize at the cell surface but is mainly released in the culture media in a fully glycosylated soluble form. The cellular behavior of anchorless PrP explains why PrP Delta GPI Tg mice can be infected but do not show the classical signs of the disorder, thus indicating that the plasma membrane localization of PrP(C) and/or of the converted scrapie form might be necessary for the development of a symptomatic disease.     

Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

Amino acid conditions near the GPI anchor attachment site of prion protein for the conversion and the GPI anchoring

Prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored protein, and the C-terminal GPI anchor signal sequence (GPI-SS) of PrP is cleaved before GPI anchoring. However, mutations near the GPI anchor attachment site (the ω site) in the GPI-SS have been recognized in human genetic prion diseases. Moreover, the ω site of PrP has not been identified except hamster, though it is known that amino acid restrictions are very severe at the ω and ω + 2 sites in other GPI-anchored proteins. To investigate the effect of mutations near the ω site of PrP on the conversion and the GPI anchoring, and to discover the ω site of murine PrP, we systematically created mutant murine PrP with all possible single amino acid substitutions at every amino acid residue from codon 228 to 240. We transfected them into scrapie-infected mouse neuroblastoma cells and examined the conversion efficiencies and the GPI anchoring of each mutant PrP. Mutations near the ω site altered the conversion efficiencies and the GPI anchoring efficiencies. Especially, amino acid restrictions for the conversion and the GPI anchoring were severe at codons 230 and 232 in murine PrP, though they were less severe than in other GPI-anchored proteins. Only the mutant PrPs presented on a cell surface via a GPI anchor were conversion competent. The present study shows that mutations in the GPI-SS can affect the GPI anchoring and the conversion efficiency of PrP. We clarified for the first time the ω site of murine PrP and the amino acid conditions near the ω site for the conversion as well as GPI anchoring.     

Internalization of mammalian fluorescent cellular prion protein and N-terminal deletion mutants in living cells

The cellular prion protein (PrP(c)) is a glycosylphosphatidylinositol (GPI)-anchored plasma membrane protein whose conformational altered forms (PrP(sc)) are known to cause neurodegenerative diseases in mammals. In order to investigate the intracellular traffic of mammalian PrP(c) in living cells, we have generated a green fluorescent protein (GFP) tagged version of PrP(c). The recombinant protein was properly anchored at the cell surface and its distribution pattern was similar to that of the endogenous PrP(c), with labeling at the plasma membrane and in an intracellular perinuclear compartment. Comparison of the steady-state distribution of GFP-PrP(c) and two N-terminal deletion mutants (Delta32-121 and Delta32-134), that cause neurological symptoms when expressed in PrP knockout mice, was carried out. The mutant proteins accumulated in the plasma membrane at the expense of decreased labeling in the perinuclear region when compared with GFP-PrP(c). In addition, GFP-PrP(c), but not the two mutants, internalized from the plasma membrane in response to Cu2+ treatment and accumulated at a perinuclear region in SN56 cells. Our data suggest that GFP-PrP(c) can be used to follow constitutive and induced PrP(c) traffic in living cells.     

Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity

According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.     

The Fate of PrP GPI‐Anchor Signal Peptide is Modulated by P238S Pathogenic Mutation

Glycosylphosphatidylinositol (GPI)‐anchored proteins are localized to the plasma membrane via a C‐terminally linked GPI anchor. The GPI anchor is added concomitantly to the cleavage of the carboxy‐terminal GPI‐anchor signal sequence, thereby causing the release of a C‐terminal hydrophobic peptide, whose fate has not yet been investigated. Here we followed the fate of the GPI‐attachment signal of the prion protein (PrP), a protein implicated in various types of transmissible neurodegenerative spongiform encephalopathies (TSE). The PrP GPI‐anchor signal sequence shows a remarkable and unusual degree of conservation across the species and contains two point mutations (M232R/T and P238S) that are responsible for genetic forms of prion disorders. We show that the PrP GPI‐anchor signal peptide (SP), but not the one from an unrelated GPI‐anchored protein (folate receptor), undergoes degradation via the proteasome. Moreover, the P238S point mutation partially protects the PrP GPI‐anchor SP from degradation. Our data provide the first attempt to address the fate of a GPI‐anchor SP and identify a role for the P238S mutation, suggesting the possibility that the PrP GPI‐anchor SP could play a role in neurodegenerative prion diseases.      

Dual mechanisms for shedding of the cellular prion protein

The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.     

Determinants of the in vivo folding of the prion protein. A bipartite function of helix 1 in folding and aggregation

Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases. We analyzed wild type PrP in comparison with different PrP mutants and identified determinants of the in vivo folding pathway of PrP. The complete N terminus of PrP including the putative transmembrane domain and the first beta-strand could be deleted without interfering with PrP maturation. Helix 1, however, turned out to be a major determinant of PrP folding. Disruption of helix 1 prevented attachment of the glycosylphosphatidylinositol (GPI) anchor and the formation of complex N-linked glycans; instead, a high mannose PrP glycoform was secreted into the cell culture supernatant. In the absence of a C-terminal membrane anchor, however, helix 1 induced the formation of unglycosylated and partially protease-resistant PrP aggregates. Moreover, we could show that the C-terminal GPI anchor signal sequence, independent of its role in GPI anchor attachment, mediates core glycosylation of nascent PrP. Interestingly, conversion of high mannose glycans to complex type glycans only occurred when PrP was membrane-anchored. Our study indicates a bipartite function of helix 1 in the maturation and aggregation of PrP and emphasizes a critical role of a membrane anchor in the formation of complex glycosylated PrP.     

Neurodegeneration induced by clustering of sialylated glycosylphosphatidylinositols of prion proteins

The transmissible spongiform encephalopathies, more commonly known as the prion diseases, are associated with the production and aggregation of disease-related isoforms of the prion protein (PrP(Sc)). The mechanisms by which PrP(Sc) accumulation causes neurodegeneration in these diseases are poorly understood. In cultured neurons, the addition of PrP(Sc) alters cell membranes, increasing cholesterol, activating cytoplasmic phospholipase A(2) (cPLA(2)), and triggering synapse damage. These effects of PrP(Sc) are dependent upon its glycosylphosphatidylinositol (GPI) anchor, suggesting that it is the increased density of GPIs that occurs following the aggregation of PrP(Sc) molecules that triggers neurodegeneration. This hypothesis was supported by observations that cross-linkage of the normal cellular prion protein (PrP(C)) also increased membrane cholesterol, activated cPLA(2), and triggered synapse damage. These effects were not seen after cross-linkage of Thy-1, another GPI-anchored protein, and were dependent on the GPI anchor attached to PrP(C) containing two acyl chains and sialic acid. We propose that the aggregation of PrP(Sc), or the cross-linkage of PrP(C), causes the clustering of sialic acid-containing GPI anchors at high densities, resulting in altered membrane composition, the pathological activation of cPLA(2), and synapse damage.     

Copper binding and conformation of the N-terminal octarepeats of the prion protein in the presence of DPC micelles as membrane mimetic

The prion protein is usually pictured as globular structured C-terminal domain that is linked to an extended flexible N-terminal tail. However, in its physiological form, it is a glycoprotein tethered to the cell surface via a C-terminal GPI anchor. The low solubility of PrP even without GPI anchor and its strong tendency for aggregation has forced most structural investigations to be performed at low pH and mostly with N-terminally truncated variants. In the present study, we have used a synthetic peptide related to the PrP tetra-octarepeat region, i.e., the sequence (Pro-His-Gly-Gly-Gly-Trp-Gly-Gln)(4), for NMR structural analysis of its preferred conformation in DPC micelles as membrane mimic. Well-defined and identical loops are observed between the four octarepeats that are linked by flexible Gly-Gly-Gly sequences. Interaction with the micelles is mainly through the tryptophan residues that appear to act as anchors. Copper binding to the peptide in the presence of DPC micelles revealed marked conformational rearrangements although binding to the micelles is preserved. Interestingly, titration experiments point to cooperative effects for the four binding sites. A destabilization of the DPC micelles by the peptide parallels the destabilizing effect of the prion protein on membranes so that the octarepeat region appears to be very membrane-active. How the physico-chemical properties reported here are linked to the function and significance of the prion protein remains a puzzle as long as the functional mechanism of the prion protein is not precisely elucidated. Nevertheless, our results emphasize the strong influence of the (membrane) environment on the PrP properties.     

Monoacylated cellular prion protein modifies cell membranes, inhibits cell signaling, and reduces prion formation

Prion diseases occur following the conversion of the cellular prion protein (PrP(C)) into a disease related, protease-resistant isoform (PrP(Sc)). In these studies, a cell painting technique was used to introduce PrP(C) to prion-infected neuronal cell lines (ScGT1, ScN2a, or SMB cells). The addition of PrP(C) resulted in increased PrP(Sc) formation that was preceded by an increase in the cholesterol content of cell membranes and increased activation of cytoplasmic phospholipase A(2) (cPLA(2)). In contrast, although PrP(C) lacking one of the two acyl chains from its glycosylphosphatidylinositol (GPI) anchor (PrP(C)-G-lyso-PI) bound readily to cells, it did not alter the amount of cholesterol in cell membranes, was not found within detergent-resistant membranes (lipid rafts), and did not activate cPLA(2). It remained within cells for longer than PrP(C) with a conventional GPI anchor and was not converted to PrP(Sc). Moreover, the addition of high amounts of PrP(C)-G-lyso-PI displaced cPLA(2) from PrP(Sc)-containing lipid rafts, reduced the activation of cPLA(2), and reduced PrP(Sc) formation in all three cell lines. In addition, ScGT1 cells treated with PrP(C)-G-lyso-PI did not transmit infection following intracerebral injection to mice. We propose that that the chemical composition of the GPI anchor attached to PrP(C) modified the local membrane microenvironments that control cell signaling, the fate of PrP(C), and hence PrP(Sc) formation. In addition, our observations raise the possibility that pharmacological modification of GPI anchors might constitute a novel therapeutic approach to prion diseases.     

Prion protein-related proteins from zebrafish are complex glycosylated and contain a glycosylphosphatidylinositol anchor

A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.     

Ablation of the metal ion-induced endocytosis of the prion protein by disease-associated mutation of the octarepeat region.

The neurodegenerative spongiform encephalopathies, or prion diseases, are characterized by the conversion of the normal cellular form of the prion protein PrP(C) to a pathogenic form, PrP(Sc) [1]. There are four copies of an octarepeat PHGG(G/S)WGQ that specifically bind Cu(2+) ions within the N-terminal half of PrP(C) [2--4]. This has led to proposals that prion diseases may, in part, be due to abrogation of the normal cellular role of PrP(C) in copper homeostasis [5]. Here, we show that murine PrP(C) is rapidly endocytosed upon exposure of neuronal cells to physiologically relevant concentrations of Cu(2+) or Zn(2+), but not Mn(2+). Deletion of the four octarepeats or mutation of the histidine residues (H68/76 dyad) in the central two repeats abolished endocytosis, indicating that the internalization of PrP(C) is governed by metal binding to the octarepeats. Furthermore, a mutant form of PrP that contains nine additional octarepeats and is associated with familial prion disease [6] failed to undergo Cu(2+)-mediated endocytosis. For the first time, these results provide evidence that metal ions can promote the endocytosis of a mammalian prion protein in neuronal cells and that neurodegeneration associated with some prion diseases may arise from the ablation of this function due to mutation of the octarepeat region.     

The role of the prion protein membrane anchor in prion infection

In transmissible spongiform encephalopathies (TSE or prion diseases) such as sheep scrapie, bovine spongiform encephalopathy and human Creutzfeldt-Jakob disease, normally soluble and protease-sensitive prion protein (PrP-sen or PrPC) is converted to an abnormal, insoluble and protease-resistant form termed PrP-res or PrPSc. PrP-res/PrPSc is believed to be the main component of the prion, the infectious agent of the TSE/prion diseases. Its precursor, PrP-sen, is anchored to the cell surface at the C-terminus by a co-translationally added glycophosphatidyl-inositol (GPI) membrane anchor which can be cleaved by the enzyme phosphatidyl-inositol specific phospholipase (PIPLC). The GPI anchor is also present in PrP-res, but is inaccessible to PIPLC digestion suggesting that conformational changes in PrP associated with PrP-res formation have blocked the PIPLC cleavage site. Although the GPI anchor is present in both PrP-sen and PrP-res, its precise role in TSE diseases remains unclear primarily because there are data to suggest that it both is and is not necessary for PrP-res formation and prion infection.     

Synthesis and structural characterization of a mimetic membrane-anchored prion protein

During pathogenesis of transmissible spongiform encephalopathies (TSEs) an abnormal form (PrP(Sc)) of the host encoded prion protein (PrP(C)) accumulates in insoluble fibrils and plaques. The two forms of PrP appear to have identical covalent structures, but differ in secondary and tertiary structure. Both PrP(C) and PrP(Sc) have glycosylphospatidylinositol (GPI) anchors through which the protein is tethered to cell membranes. Membrane attachment has been suggested to play a role in the conversion of PrP(C) to PrP(Sc), but the majority of in vitro studies of the function, structure, folding and stability of PrP use recombinant protein lacking the GPI anchor. In order to study the effects of membranes on the structure of PrP, we synthesized a GPI anchor mimetic (GPIm), which we have covalently coupled to a genetically engineered cysteine residue at the C-terminus of recombinant PrP. The lipid anchor places the protein at the same distance from the membrane as does the naturally occurring GPI anchor. We demonstrate that PrP coupled to GPIm (PrP-GPIm) inserts into model lipid membranes and that structural information can be obtained from this membrane-anchored PrP. We show that the structure of PrP-GPIm reconstituted in phosphatidylcholine and raft membranes resembles that of PrP, without a GPI anchor, in solution. The results provide experimental evidence in support of previous suggestions that NMR structures of soluble, anchor-free forms of PrP represent the structure of cellular, membrane-anchored PrP. The availability of a lipid-anchored construct of PrP provides a unique model to investigate the effects of different lipid environments on the structure and conversion mechanisms of PrP.     

Intercellular transfer of the cellular prion protein

The cellular prion protein (PrP(C)) is a glycosylphosphatidylinositol (GPI)-anchored protein. We investigated whether PrP(C) can move from one cell to another cell in a cell model. Little PrP(C) transfer was detected when a PrP(C) expressing human neuroblastoma cell line was cultured with the human erythroleukemia cells IA lacking PrP(C). Efficient transfer of PrP(C) was detected with the presence of phorbol 12-myristate 13-acetate, an activator of protein kinase C. Maximum PrP(C) transfer was observed when both donor and recipient cells were activated. Furthermore, PrP(C) transfer required the GPI anchor and direct cell to cell contact. However, intercellular protein transfer is not limited to PrP(C), another GPI-anchored protein, CD90, also transfers from the donor cells to acceptor cells after cellular activation. Therefore, this transfer process is GPI-anchor and cellular activation dependent. These findings suggest that the intercellular transfer of GPI-anchored proteins is a regulated process, and may have implications for the pathogenesis of prion disease.     

Biosynthesis of the vacuolar H+-ATPase accessory subunit Ac45 in Xenopus pituitary.

Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.