Cytoplasmic Face Of Membrane Research Articles

Wnt Isoform-Specific Interactions with Coreceptor Specify Inhibition or Potentiation of Signaling by LRP6 Antibodies

β-catenin-dependent Wnt signaling is initiated as Wnt binds to both the receptor FZD and coreceptor LRP5/6, which then assembles a multimeric complex at the cytoplasmic membrane face to recruit and inactivate the kinase GSK3. The large number and sequence diversity of Wnt isoforms suggest the possibility of domain-specific ligand-coreceptor interactions, and distinct binding sites on LRP6 for Wnt3a and Wnt9b have recently been identified in vitro. Whether mechanistically different interactions between Wnts and coreceptors might mediate signaling remains to be determined. It is also not clear whether coreceptor homodimerization induced extracellularly can activate Wnt signaling, as is the case for receptor tyrosine kinases. We generated monoclonal antibodies against LRP6 with the unexpected ability to inhibit signaling by some Wnt isoforms and potentiate signaling by other isoforms. In cell culture, two antibodies characterized further show reciprocal activities on most Wnts, with one antibody antagonizing and the other potentiating. We demonstrate that these antibodies bind to different regions of LRP6 protein, and inhibition of signaling results from blocking Wnt binding. Antibody-mediated dimerization of LRP6 can potentiate signaling only when a Wnt isoform is also able to bind the complex, presumably recruiting FZD. Endogenous autocrine Wnt signaling in different tumor cell lines can be either antagonized or enhanced by the LRP6 antibodies, indicating expression of different Wnt isoforms. As anticipated from the roles of Wnt signaling in cancer and bone development, antibody activities can also be observed in mice for inhibition of tumor growth and in organ culture for enhancement of bone mineral density. Collectively, our results indicate that separate binding sites for different subsets of Wnt isoforms determine the inhibition or potentiation of signaling conferred by LRP6 antibodies. This complexity of coreceptor-ligand interactions may allow for differential regulation of signaling by Wnt isoforms during development, and can be exploited with antibodies to differentially manipulate Wnt signaling in specific tissues or disease states.

Evolution of the spectrin-based membrane skeleton

A group of four proteins – spectrin, ankyrin, 4.1 and adducin – evolved with the metazoa. These membrane-cytoskeletal proteins cross-link actin on the cytoplasmic face of plasma membranes and link a variety of transmembrane proteins to the cytoskeleton. In this paper, the evolution of these proteins is analysed. Genomics indicate that spectrin was the first to appear, since the genome of the choanoflagellate Monosiga brevicolis contains genes for α, β and βH spectrin. This organism represents a lineage of free-living and colonial protists from which the metazoa are considered to have diverged. This indicates that spectrin emerged in evolution before the animals. Simple animals such as the placozoan Trichoplax adherens also contain recognizable precursors of 4.1, ankyrin and adducin, but these could probably not bind spectrin. Ankyrin and adducin seem to have acquired spectrin-binding activity with the appearance of tissues since they appear to have largely the same domain structure in all eumetazoa. 4.1 was adapted more recently, with the emergence of the vertebrates, to bind spectrin and promote its interaction with actin. A simple hypothesis is that spectrin was prerequisite (but not sufficient) for animal life; that spectrin interaction with ankyrin and adducin was required for evolution of major tissues; and that 4.1 acquired a spectrin-actin binding activity as animal size increased with the appearance of vertebrates. The spectrin/ankyrin/adducin/4.1 complex represents a remarkable system that underpins animal life; it has been adapted to many different functions at different times during animal evolution.

Structural Characterization of Apical Membrane Antigen 1 (AMA1) from Toxoplasma gondii

Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.

Protein domains, catalytic activity, and subcellular distribution of mouse NTE-related esterase

A mammalian family of lipid hydrolases, designated "patatin-like phospholipase domain containing (PNPLA)" recently has attracted attention. NTE-related esterase (NRE) as a member of PNPLA is an insulin-regulated lysophospholipase with homology to neuropathy target esterase (NTE). Mouse NRE (mNRE) has a predicted amino-terminal transmembrane region (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. In the current study, we described the expression of green fluorescent protein (GFP)-tagged constructs of mNRE and mutant proteins lacking the specific protein domains. Esterase assays indicated that neither the TM nor R-domain was essential for mNRE esterase activity, but the TM significantly contributed to its activity. Subcellular distribution showed that mNRE was anchored in ER via its TM domain and that its C-domain was associated with ER. Furthermore, experiments involving proteinase treatment revealed that most of mNRE molecule was exposed on the cytoplasmic face of ER membranes. Collectively, our results for the first time revealed the protein domains, catalytic activity, and subcellular location of mNRE and a simplified model for mNRE was proposed.

2-Bromopalmitate and 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one inhibit DHHC-mediated palmitoylation in vitro

Pharmacologic approaches to studying palmitoylation are limited by the lack of specific inhibitors. Recently, screens have revealed five chemical classes of small molecules that inhibit cellular processes associated with palmitoylation (Ducker, C. E., L. K. Griffel, R. A. Smith, S. N. Keller, Y. Zhuang, Z. Xia, J. D. Diller, and C. D. Smith. 2006. Discovery and characterization of inhibitors of human palmitoyl acyltransferases. Mol. Cancer Ther. 5: 1647-1659). Compounds that selectively inhibited palmitoylation of N-myristoylated vs. farnesylated peptides were identified in assays of palmitoyltransferase activity using cell membranes. Palmitoylation is catalyzed by a family of enzymes that share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain. In this study, we evaluated the ability of these inhibitors to reduce DHHC-mediated palmitoylation using purified enzymes and protein substrates. Human DHHC2 and yeast Pfa3 were assayed with their respective N-myristoylated substrates, Lck and Vac8. Human DHHC9/GCP16 and yeast Erf2/Erf4 were tested using farnesylated Ras proteins. Surprisingly, all four enzymes showed a similar profile of inhibition. Only one of the novel compounds, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one [Compound V (CV)], and 2-bromopalmitate (2BP) inhibited the palmitoyltransferase activity of all DHHC proteins tested. Hence, the reported potency and selectivity of these compounds were not recapitulated with purified enzymes and their cognate lipidated substrates. Further characterization revealed both compounds blocked DHHC enzyme autoacylation and displayed slow, time-dependent inhibition but differed with respect to reversibility. Inhibition of palmitoyltransferase activity by CV was reversible, whereas 2BP inhibition was irreversible.

Protein aging: Truncation of aquaporin 0 in human lens regions is a continuous age-dependent process

The human lens is ideal for the study of macromolecular aging because cells in the centre, along with their constituent proteins, are present for our entire lives. We examined the major membrane protein, aquaporin 0 (AQP0), in regions of the lens formed at different times during our lifespan, to determine if similar changes could be detected and if they were progressive. Membrane fractions from three concentric lens regions were examined by SDS-PAGE coupled with densitometry, and Western blotting, to assess the time course of truncation. The overall extent of modification was also examined by MALDI mass spectrometry of the undigested proteins. In all regions, AQP0 became progressively more truncated, specifically by the loss of a 2 kDa intracellular C-terminal peptide. The proteolysis increased steadily in all regions such that half of the AQP0 in the barrier region (that part of the lens formed immediately after birth) had been cleaved by age 40–50. MALDI mass spectrometry revealed that in all regions, AQP0 not only was shortened, it also became progressively more heterogeneous with age. Since the lens interior is devoid of active enzymes, it is very likely that the cleavage of AQP0 is chemically induced. We speculate that the loss of this C-terminal peptide ‘spacer’ may allow occlusion of AQP0 pores on the cytoplasmic face of the fibre cell membranes. Once a significant proportion of AQP0 has been cleaved, this occlusion may contribute to the formation of the lens permeability barrier that develops at middle age.

Novel components of the Apicomplexan moving junction reveal conserved and coccidia-restricted elements

Apicomplexan parasites generally invade their host cells by anchoring the parasite to the host membrane through a structure called the moving junction (MJ). This MJ is also believed to sieve host proteins from the nascent parasitophorous vacuole membrane, which likely protects the pathogen from lysosomal destruction. Previously identified constituents of the Toxoplasma MJ have orthologues in Plasmodium, indicating a conserved structure throughout the Apicomplexa. We report here two novel MJ proteins, RON5 and RON8. While RON5 is conserved in Plasmodium, RON8 appears restricted to the coccidia. RON8, which is likely essential, co-immunoprecipitates RON5 and known MJ proteins from extracellular parasites, indicating that a preformed complex exists within the parasites. Upon secretion, we show that RON8 within the MJ localizes to the cytoplasmic face of the host plasma membrane. To examine interactions between RON8 and the host cell, we expressed RON8 in mammalian cells and show that it targets to its site of action at the periphery in a manner dependent on the C-terminal portion of the protein. The discovery of RON5 and RON8 provides new insight into conserved and unique elements of the MJ, furthering our understanding of how the MJ contributes to the intricate mechanism of Apicomplexan invasion.

The plant-specific TFIIB-related protein, pBrp, is a general transcription factor for RNA polymerase I

TFIIB and BRF are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo, and the occupancy was proportional to de novo 18S rRNA synthesis. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro, and the binding site was identified at a proximal downstream region of the transcription start point. alpha-Amanitin-resistant transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate and co-localize with the RNA polymerase I subunit, CmRPA190, in the cell. Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

ZFPL1, a novel ring finger protein required for cis-Golgi integrity and efficient ER-to-Golgi transport

The Golgi apparatus occupies a central position within the secretory pathway, but the molecular mechanisms responsible for its assembly and organization remain poorly understood. We report here the identification of zinc finger protein-like 1 (ZFPL1) as a novel structural component of the Golgi apparatus. ZFPL1 is a conserved and widely expressed integral membrane protein with two predicted zinc fingers at the N-terminus, the second of which is a likely ring domain. ZFPL1 directly interacts with the cis-Golgi matrix protein GM130. Depletion of ZFPL1 results in the accumulation of cis-Golgi matrix proteins in the intermediate compartment (IC) and the tubulation of cis-Golgi and IC membranes. Loss of ZFPL1 function also impairs cis-Golgi assembly following brefeldin A washout and slows the rate of cargo trafficking into the Golgi apparatus. Effects upon Golgi matrix protein localization and cis-Golgi structure can be rescued by wild-type ZFPL1 but not mutants defective in GM130 binding. Together, these data suggest that ZFPL1 has an important function in maintaining the integrity of the cis-Golgi and that it does so through interactions with GM130.

Reduced redox potential of the cytosol is important for African swine fever virus capsid assembly and maturation

Assembly of African swine fever virus (ASFV) involves the transfer of the major capsid protein, p73, from the cytosol onto the cytoplasmic face of endoplasmic reticulum-derived membranes. During this process, the folding of p73 is dependent upon transient association with a specific viral chaperone, CAP80. The cell cytoplasm maintains high concentrations of reduced glutathione, leading to a reducing environment. Here, the effects of redox environment on the assembly of ASFV have been studied. Diamide, which oxidizes the cell cytosol, slowed the folding of p73 and prevented release from CAP80 and subsequent binding of p73 to membranes. Similarly, cell oxidation slowed the assembly of p73 molecules already bound to membranes into virus capsid precursors. Interestingly, addition of oxidized glutathione to newly assembled virus capsid precursors in vitro led to disassembly; however, virus particles released from cells were resistant to oxidized glutathione. These data show that assembly of ASFV requires the reducing environment that prevails in the cytosol, but as the virus matures, it becomes resistant to oxidation, possibly indicating preparation for release from the cell.

Cellular and subcellular localization of paralemmin-1, a protein involved in cell shape control, in the rat brain, adrenal gland and kidney

Paralemmin-1 is a phosphoprotein, lipid-anchored to the cytoplasmic face of membranes and implicated in plasma membrane dynamics and cell process formation. We report an immunoperoxidase histochemical analysis of the cellular and subcellular localization of paralemmin-1 in the rat tissues where its expression is highest: the brain, the adrenal gland and the kidney. Paralemmin-1 is detected throughout the brain, in neuronal perikarya, axons and dendrites including dendritic spines and also in glial processes. In the adrenal gland, paralemmin-1 is highly expressed in the medulla. The kidney displays a pattern of differential paralemmin-1 expression in various structures and cell types, with high concentrations in cells of the parietal epithelium of Bowman's capsule, intermediate tubules, distal tubules and principal cells of outer medullary collecting ducts. Mosaics of paralemmin-positive and paralemmin-negative cells are observed in proximal tubules, the parietal epithelium of Bowman's capsule and the endothelium of many blood vessels. Plasma membrane association in epithelia is often polarized: paralemmin-1 concentrates at the apical membranes of adrenal chromaffin cells, but at the basolateral plasma membranes of proximal and distal tubule cells in the kidney. Paralemmin-1 immunoreactivity exhibits a spotted pattern and can be seen both at plasma membranes and within the cytoplasm, where it is often associated with endomembranes. This discontinuous distribution and the detergent extraction properties of paralemmin-1 suggest an association with lipid microdomains. The findings are consistent with a role for paralemmin-1 in the formation and stabilization of plasma membrane elaborations, in neurons as well as in other cell types.

The ruffled border and attachment regions of the apposing membrane of resorbing osteoclasts as visualized from the cytoplasmic face of the membrane

The aim of our present research was to visualize how the plasma membrane is modified and how the cytoskeleton interacts with the attachment and ruffled border regions of resorbing osteoclasts. In order to view the surface modification of membranes and associated cytoskeleton, we employed the method of cell-shearing combined with quick-freezing and rotary replication to expose and replicate an extensive area of the cytoplasmic face of the surface membrane of osteoclasts in contact with synthetic apatite as a substratum. The membrane apposed to the apatite was composed of three different domains: the attachment zone, ruffled border and the remainder. In the attachment zone, a highly organized actin filament network formed dot-shaped, F-actin rich adhesion sites, so-called podosomes, and the actin ring. The cytoskeletal filament of podosomes and actin ring appeared to be in direct contact with the cytoplasmic surface of the underlying membrane. Within the actin ring, individually recognizable podosomes were well preserved, which indicates that the actin ring was probably derived from the fusion of podosomes. After shearing at the ruffled border region, the ruffled border projections and membrane regions among the projections were left behind. These ruffled border projections contained the cytoskeletal network. These actin networks also appeared to be in direct contact with the inner side of the ruffled border membrane or in contact with it via membrane-associated particles. At the basal portion of the ruffled border, numerous clathrin-coated patches or pits were well preserved. Deeper clathrin-coated pits and vesicles were also found, which indicates an active site for receptor-mediated endocytotic events. Clathrin sheets were also observed in the cell periphery outside of the actin ring. This type of clathrin sheets adhered to the apatite substrate, but was not anchored to the actin microfilaments. Our study thus clearly visualized the interaction between the cytoskeletal filaments and the underlying membrane at the ruffled border, attachment zone and podosome in osteoclasts cultured on apatitepellets.

Cloning and Expression of Sheep Renal K-CI Cotransporter-1

Sheep K-Cl cotransporter-1(shKCC1) cDNA was cloned from kidney by RT-PCR with an open reading frame of 3258 base pairs exhibiting 92%, 90%, 88% and 87% identity with pig, rabbit and human, rat and mouse KCC1 cDNAs, respectively, encoding a ñ122 kDa polypeptide of 1086-amino acids. Hydropathy analysis reveals the familiar KCC1 topology with 12 transmembrane domains (TMDs) and the hydrophilic NH₂-terminal (NTD) and COOH-terminal (CTD) domains both at the cytoplasmic membrane face. However, shKCC1 has two rather than one large extracellular loops (ECL): ECL3 between TMDs 5 and 6, and ECL6, between TMDs 11 and 12. The translated shKCC1 protein differs in 12 amino acid residues from other KCC1s, mainly within the NTD, ECL3, ICL4, ECL6, and CTD. Notably, a tyrosine residue at position 996 replaces aspartic acid conserved in all other species. Human embryonic kidney (HEK293) cells and mouse NIH/3T3 fibroblasts, transiently transfected with shKCCI-cDNA, revealed the glycosylated ñ150 kDa proteins by Western blots and positive immunofluorescence-staining with polyclonal rabbit anti-ratKCC1 antibodies. ShKCC1 was functionally expressed in NIH/3T3 cells by an elevated basal Cl-dependent K influx measured with Rb as K-congener that was stimulated three-fold by the KCC-activator N-ethylmaleimide.

Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells

Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB(10463) between Leu(543) and Gly(544) and the naturally occurring variant TcdB(8864) between Leu(544) and Gly(545). The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB(10463)) and 62 761 Da (TcdB(8864))] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB(10463)) and 206 245 Da (TcdB(8864))] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

Redistribution of cellular and herpes simplex virus proteins from the trans-golgi network to cell junctions without enveloped capsids.

Herpes simplex virus (HSV) and other alphaherpesviruses assemble enveloped virions in the trans-Golgi network (TGN) or endosomes. Enveloped particles are formed when capsids bud into TGN/endosomes and virus particles are subsequently ferried to the plasma membrane in TGN-derived vesicles. Little is known about the last stages of virus egress from the TGN/endosomes to cell surfaces except that the HSV directs transport of nascent virions to specific cell surface domains, i.e., epithelial cell junctions. Previously, we showed that HSV glycoprotein gE/gI accumulates extensively in the TGN at early times after infection and also when expressed without other viral proteins. At late times of infection, gE/gI and a cellular membrane protein, TGN46, were redistributed from the TGN to epithelial cell junctions. We show here that gE/gI and a second glycoprotein, gB, TGN46, and another cellular protein, carboxypeptidase D, all moved to cell junctions after infection with an HSV mutant unable to produce cytoplasmic capsids. This redistribution did not involve L particles. In contrast to TGN membrane proteins, several cellular proteins that normally adhere to the cytoplasmic face of TGN, Golgi, and endosomal membranes remained primarily dispersed throughout the cytoplasm. Therefore, cellular and viral membrane TGN proteins move to cell junctions at late times of HSV infection when the production of enveloped particles is blocked. This is consistent with the hypothesis that there are late HSV proteins that reorganize or redistribute TGN/endosomal compartments to promote virus egress and cell-to-cell spread.

Omega-3 fatty acids in colorectal cancer prevention.

Omega-3 fatty acids in colorectal cancer prevention.

Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease.

Mutations in ALS2, carrying three putative guanine exchange factor (GEF) domains, are causative for a juvenile, autosomal recessive form of amyotrophic lateral sclerosis (ALS), primary lateral sclerosis, and infantile-ascending hereditary spastic paralysis. Endogenous ALS2 is shown here to be enriched in nervous tissue and to be peripherally bound to the cytoplasmic face of endosomal membranes, an association that requires the amino-terminal "RCC1 (regulator of chromatin condensation)-like" GEF domain. Disease-causing mutants and a naturally truncated isoform of ALS2 are shown to be rapidly degraded when expressed in cultured human cells, including lymphocytes derived from patients with ALS2 mutations. Thus, mutations in the ALS2 gene linked to early-onset motor neuron disease uniformly produce loss of activity through decreased protein stability of this endosomal GEF.

Binding partners for the UL11 tegument protein of herpes simplex virus type 1.

The product of the U(L)11 gene of herpes simplex virus type 1 (HSV-1) is a 96-amino-acid tegument protein that accumulates on the cytoplasmic face of internal membranes. Although it is thought to be important for nucleocapsid envelopment and egress, the actual function of this protein is unknown. Previous studies focused on the characterization of sequence elements within the UL11 protein that function in membrane binding and trafficking to the Golgi apparatus. Binding was found to be mediated by two fatty acyl groups (myristate and palmitate), while an acidic cluster and a dileucine motif were identified as being important for the recycling of UL11 from the plasma membrane to the Golgi apparatus. The goal of the experiments described here was to identify and characterize binding partners (viral or cellular) of UL11. Using both immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we identified a 40-kDa protein that specifically associates with UL11 from infected Vero cells. Mutational analyses revealed that the acidic cluster and the dileucine motif are required for this association, whereas the entire second half of UL11 is not. In addition, UL11 homologs from pseudorabies and Marek's disease herpesviruses were also found to be capable of binding to the 40-kDa protein from HSV-1-infected cells, suggesting that the interaction is conserved among alphaherpesviruses. Purification and analysis of the 40-kDa protein by mass spectrometry revealed that it is the product of the U(L)16 gene, a virion protein reported to be involved in nucleocapsid assembly. Cells transfected with a UL16-green fluorescent protein expression vector produced a protein that was of the expected size, could be pulled down with GST-UL11, and accumulated in a Golgi-like compartment only when coexpressed with UL11, indicating that the interaction does not require any other viral products. These data represent the first steps toward elucidating the network of tegument proteins that UL11 links to membranes.

Protein Domains, Catalytic Activity, and Subcellular Distribution of Neuropathy Target Esterase in Mammalian Cells

Neuropathy target esterase (NTE), the human homologue of a protein required for brain development in Drosophila, has a predicted amino-terminal transmembrane helix (TM), a putative regulatory (R) domain, and a hydrophobic catalytic (C) domain. Here we describe the expression, in COS cells, of green fluorescent protein-tagged constructs of NTE and mutant proteins lacking the TM or the R- or C-domains. Esterase assays and Western blots of particulate and soluble fractions indicated that neither the TM nor R-domain is essential for NTE catalytic activity but that this activity requires membrane association to which the TM, R-, and C-domains all contribute. Experiments involving proteinase treatment revealed that most of the NTE molecule is exposed on the cytoplasmic face of membranes. In cells expressing a moderate level of NTE and all cells expressing DeltaC-NTE, fluorescence was distributed in an endoplasmic reticulum (ER)-like pattern. Cells expressing high levels of NTE showed aberrant distribution of ER marker proteins and accumulation of NTE on the cytoplasmic surface of ER-derived tubuloreticular aggregates. Deformation of the ER was also seen in cells expressing DeltaR-NTE or enzymatically inactive S966A-NTE but not DeltaTM-NTE. The data suggest that NTE is anchored in the ER via its TM, that its R- and C-domains also interact with the cytoplasmic face of the ER, and that overexpression of NTE causes ER aggregation via intermolecular association of its C-domains.

Biochemical and immunocytochemical characterization of calsenilin in mouse brain

Mutations in the presenilin 1 and 2 genes cause the majority of early onset familial forms of Alzheimer’s disease. Here we describe the biochemical and immunohistochemical characterization of calsenilin, a novel calcium binding protein that we have previously shown to interact with presenilins 1 and 2, in mouse brain. The co-immunoprecipitation of endogenous calsenilin and presenilin 1 demonstrates that these proteins are physiologic binding partners. Although calsenilin has been predicted to be a soluble protein, we have found that the majority of it is tightly associated with the cytoplasmic face of intracellular membranes and that it can only be dissociated using harsh treatments such as urea. In addition, we have demonstrated that calsenilin is a developmentally regulated protein that is mainly present in the brain, where it localizes to both the hippocampus and cerebellum. Calsenilin staining co-localized with the somatodendritic marker microtubule-associated protein-2 primarily in the granular cell layer of the cerebellum, indicating that calsenilin expression is primarily neuronal. In primary cultured neurons, calsenilin immunoreactivity was observed in cell bodies as well as in some neuronal processes. Co-localization experiments using specific axonal and dendritic markers indicate that these processes were mainly axonal in nature, although a smaller subset of dendrites also appears to contain calsenilin. In summary, we have established that calsenilin and presenilin 1 can interact at physiologic levels, and that calsenilin is a developmentally regulated protein that is expressed primarily in the cerebellum and hippocampus. Although calsenilin is a soluble protein, it is tightly associated with the membrane. Finally, the expression pattern of calsenilin, which is similar to that of the presenilin(s), suggests that the common locations of these two proteins provide an opportunity for physical interaction in vivo.