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.     

Subcellular localization and targeting of N-acetylglucosaminyl phosphatidylinositol de-N-acetylase, the second enzyme in the glycosylphosphatidylinositol biosynthetic pathway

The second step in glycosylphosphatidylinositol biosynthesis is the de-N-acetylation of N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) catalyzed by N-acetylglucosaminylphosphatidylinositol deacetylase (PIG-L). Previous studies of mouse thymoma cells showed that GlcNAc-PI de-N-acetylase activity is localized to the endoplasmic reticulum (ER) but enriched in a mitochondria-associated ER membrane (MAM) domain. Because PIG-L has no readily identifiable ER sorting determinants, we were interested in learning how PIG-L is localized to the ER and possibly enriched in MAM. We used HeLa cells transiently or stably expressing epitope-tagged PIG-L variants or chimeric constructs composed of elements of PIG-L fused to Tac antigen, a cell surface protein. We first analyzed the subcellular distribution of PIG-L and Glc-NAc-PI-de-N-acetylase activity and then studied the localization of Tac-PIG-L chimeras to identify sequence elements in PIG-L responsible for its subcellular localization. We show that human PIG-L is a type I membrane protein with a large cytoplasmic domain and that, unlike the result with mouse thymoma cells, both PIG-L and GlcNAc-PI-de-N-acetylase activity are uniformly distributed between ER and MAM in HeLa cells. Analyses of a series of Tac-PIG-L chimeras indicated that PIG-L contains two ER localization signals, an independent retention signal located between residues 60 and 88 of its cytoplasmic domain and another weak signal in the luminal and transmembrane domains that functions autonomously in the presence of membrane proximal residues of the cytoplasmic domain that themselves lack any retention information. We conclude that PIG-L, like a number of other ER membrane proteins, is retained in the ER through a multi-component localization signal rather than a discrete sorting motif.     

Neuropathy target esterase and phospholipid deacylation

Certain organophosphates react with the active site serine residue of neuropathy target esterase (NTE) and cause axonal degeneration and paralysis. Cloning of NTE revealed the presence of homologues in eukaryotes from yeast to man and that the protein has both a catalytic and a regulatory domain. The latter contains sequences similar to the regulatory subunit of protein kinase A, suggesting that NTE may bind cyclic AMP. NTE is tethered via an amino-terminal transmembrane segment to the cytoplasmic face of the endoplasmic reticulum. Unlike wild-type yeast, mutants lacking NTE activity cannot deacylate CDP-choline pathway-synthesized phosphatidylcholine (PtdCho) to glycerophosphocholine (GroPCho) and fatty acids. In cultured mammalian cells, GroPCho levels rise and fall, respectively, in response to experimental over-expression, and inhibition, of NTE. A complex of PtdCho and Sec14p, a yeast phospholipid-binding protein, both inhibits the rate-limiting step in PtdCho synthesis and enhances deacylation of PtdCho by NTE. While yeast can maintain PtdCho homeostasis in the absence of NTE, certain post-mitotic metazoan cells may not be able to, and some NTE-null animals have deleterious phenotypes. NTE is not required for cell division in the early mammalian embryo or in larval and pupal forms of Drosophila, but is essential for placenta formation and survival of neurons in the adult. In vertebrates, the relative importance of NTE and calcium-independent phospholipase A2 for homeostatic PtdCho deacylation in particular cell types, possible interactions of NTE with Sec14p homologues and cyclic AMP, and whether deranged phospholipid metabolism underlies organophosphate-induced neuropathy are areas which require further investigation.     

Two chimeric receptors of epidermal growth factor receptor and c-Ros that differ in their transmembrane domains have opposite effects on cell growth.

Two chimeric receptors, ER1 and ER2, were constructed. ER1 contains the extracellular and transmembrane (TM) domains derived from epidermal growth factor receptor and the cytoplasmic domain from c-Ros; ER2 is identical to ER1 except that its TM domain is derived from c-Ros. Both chimeras can be activated by epidermal growth factor and are capable of activating or phosphorylating an array of cellular signaling proteins. Both chimeras promote colony formation in soft agar with about equal efficiency. Surprisingly, ER1 inhibits while ER2 stimulates cell growth on monolayer culture. Cell cycle analysis revealed that all phases, in particular the S and G2/M phases, of the cell cycle in ER1 cells were elongated whereas G1 phase of ER2 cells was shortened threefold. Comparison of signaling pathways mediated by the two chimeras revealed several differences. Several early signaling proteins are activated or phosphorylated to a higher extent in ER1 than in ER2 cells in response to epidermal growth factor. ER1 is less efficiently internalized and remains tyrosine phosphorylated for a longer time than ER2. However, phosphorylation of the 66-kDa She protein, activation of mitogen activated protein kinase, and induction of c-fos and c-jun occur either to a lesser extent or for a shorter time in ER1 cells. Cellular protein phosphorylation patterns are also different in ER1 and ER2 cells. In particular, a 190-kDa Shc-associated protein is tyrosine phosphorylated in ER2 but not in ER1 cells. Our results indicate that the TM domains have a profound effect on the signal transduction and biological activity of those chimeric receptors. The results also imply that sustained stimulation of ER1 due to its retarded internalization apparently triggers an inhibitory response that dominantly counteracts the receptor-mediated mitogenic signals. These two chimeras, expressed at similar levels in the same cell type but having opposite effects on cell growth, provide an ideal system to study the mechanism by which a protein tyrosine kinase inhibits cell growth.     

Membrane topology and dimerization of the two subunits of the transporter associated with antigen processing reveal a three-domain structure

Presentation of peptides derived from cytosolic and nuclear proteins by MHC class I molecules requires their translocation across the membrane of the endoplasmic reticulum (ER) by a specialized ABC (ATP-binding cassette) transporter, TAP. To investigate the topology of the heterodimeric TAP complex, we constructed a set of C-terminal deletions for the TAP1 and TAP2 subunits. We identified eight and seven transmembrane (TM) segments for TAP1 and TAP2, respectively. TAP1 has both its N and C terminus in the cytoplasm, whereas TAP2 has its N terminus in the lumen of the ER. A putative TM pore consists of TM1-6 of TAP1 and, by analogy, TM1-5 of TAP2. Multiple ER-retention signals are present within this region, of which we positively identified TM1 of both TAP subunits. The N-terminal domain containing TM1-6 of TAP1 is sufficient for dimerization with TAP2. A second, independent dimerization domain, located between the putative pore and the nucleotide-binding cassette, lies within the cytoplasmic peptide-binding domains, which are anchored to the membrane via TM doublets 7/8 and 6/7 of TAP1 and TAP2, respectively. We present a model in which TAP is composed of three subdomains: a TM pore, a cytoplasmic peptide-binding pocket, and a nucleotide-binding domain.     

G protein beta2 subunit interacts directly with neuropathy target esterase and regulates its activity

Neuropathy target esterase (NTE) was identified as the primary target of organophosphate compounds that cause a delayed neuropathy with degeneration of nerve axons. NTE is a novel phospholipase B anchored to the cytoplasmic face of endoplasmic reticulum and essential for embryonic and nervous development. However, little is known about the regulation of NTE. A human fetal brain cDNA library was screened for proteins that interact with NTE, Gbeta2 and Gbeta2-like I subunits were found to be able to bind the C-terminal of NTE in yeast. The interaction of Gbeta2 and NTE was confirmed by in vivo co-immunoprecipitation analysis in COS7 cells. Furthermore, depletion of Gbeta2 by RNA interference down regulated the activity of NTE but not its expression level. In addition, the activity of NTE was down regulated by the G protein signal pathway influencing factor, pertussis toxin, treatment in vivo. These findings suggest that Gbeta2 may play a significant role in maintaining the activity of NTE.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Tomato ringspot virus proteins containing the nucleoside triphosphate binding domain are transmembrane proteins that associate with the endoplasmic reticulum and cofractionate with replication complexes

Replication of all known positive-strand RNA viruses occurs in replication complexes associated with intracellular membranes. The putative nucleoside triphosphate binding (NTB) protein of Tomato ringspot virus (ToRSV) contains a stretch of hydrophobic residues at its C terminus, suggesting that it may act as a membrane anchor for the replication complex. Anti-NTB antibodies detected two predominant proteins in membrane-enriched fractions (the 66-kDa NTB and 69-kDa NTB-VPg proteins) along with other, larger proteins. The proteins containing the NTB domain cofractionated with markers of the endoplasmic reticulum (ER) and with ToRSV-specific RNA-dependent RNA polymerase activity in sucrose gradients. ToRSV infection induced severe changes in the morphology of the ER in plants expressing an ER-targeted green fluorescent protein (ER-GFP), and proteins containing the NTB domain colocalized with ER-GFP in indirect immunofluorescence assays. The proteins containing the NTB domain have properties of integral membrane proteins. Proteinase K protection assays using purified membranes from infected plants revealed that although the central portion of the NTB domain is exposed to the cytoplasmic face of the membranes, an 8-kDa fragment, recognized by anti-VPg antibodies, is protected by the membranes. This fragment probably consists of the 3-kDa VPg and the 5-kDa stretch of hydrophobic residues at the C terminus of the NTB protein, suggesting a luminal location for the VPg in at least a portion of the molecules. These results provide evidence that proteins containing the NTB domain are transmembrane proteins associated with ER-derived membranes and support the hypothesis that one or several of the proteins containing the NTB domain anchor the replication complex to the ER.     

Intracellular Interaction of Simian Immunodeficiency Virus Gag and Env Proteins

In polarized epithelial cells, the assembly and release of human immunodeficiency virus type 1 (HIV-1) occur at the basolateral side of the plasma membrane, and the site of assembly is determined by the site of expression of the Env protein. In order to investigate whether the expression of the Env proteins exclusively in the endoplasmic reticulum (ER) can alter the site of virus assembly, we coexpressed the simian immunodeficiency virus (SIV) Gag protein and mutant SIV Env proteins having an ER retrieval signal (KKXX motif). In cells expressing the wild-type (wt) Env protein or coexpressing Env and Gag proteins, the Env protein was processed into the surface (SU) and transmembrane (TM) proteins. In contrast, in cells expressing the mutant Env proteins alone or in combination with Gag, the Env proteins were retrieved to the ER and were not proteolytically processed. Coexpression of the Gag and ER-retained mutant Env proteins resulted in a transient decrease in the release of the Gag protein into the medium, suggesting an interaction between the Gag and ER-retrieved Env proteins. Using saponin-permeabilized cells coexpressing Gag and Env proteins, we obtained further evidence for Env-Gag interaction. A monoclonal antibody specific to the SIV Gag protein was found to coimmunoprecipitate both the Gag and Env proteins. The interaction was specific, as coexpressed SIV Env proteins without the cytoplasmic tail or a chimeric HIV-1 Env proteins with the CD4 cytoplasmic tail were not coimmunoprecipitated by the Gag-specific antibody. Electron microscopic analyses indicated that assembly of virus particles occurred only at the surfaces of cells in which the Gag protein was coexpressed with either the wt or ER-retrieved mutant Env protein. These data indicate that although the Env and Gag proteins interact intracellularly, the site of assembly of SIV is not redirected to an intracellular organelle by the retrieval of the Env protein to the ER.     

The destruction box is involved in the degradation of the NTE family proteins by the proteasome

Neuropathy target esterase (NTE) and NTE-related esterase (NRE) are endoplasmic reticulum (ER) membrane-anchored proteins belonging to the NTE protein family. NTE and NRE are degraded by macroautophagy and by the ubiquitin–proteasome pathway. However, the regulation of NTE and NRE by proteasome has not been well understood. Western blotting showed that the deletion of the regulatory region of NTE and NRE led to protein accumulation compared with that of the corresponding wild-type proteins. Further, deletion and site-directed mutagenesis experiments demonstrated that the destruction (D) box was required for the proteasomal degradation of NTE and NRE. However, unlike the deletion of the regulatory region, the deletion of the D box did not affect the subcellular localisation of NTE or NRE or disrupt the ER. Moreover, the deletion of the D box or the regulatory region of NTE has similar inhibitory effects on cell growth, which are greater than those produced by the full-length NTE. Here, for the first time, we show that the D box is involved in the regulation of NTE family proteins by the proteasome but not in their subcellular localisation. In addition, these results suggest that the NTE overexpression-mediated inhibition of cell growth is related to active protein levels but not to its ER disruption effect.     

The transmembrane domain of podoplanin is required for its association with lipid rafts and the induction of epithelial-mesenchymal transition

Podoplanin is a transmembrane glycoprotein that is upregulated in cancer and was reported to induce an epithelial-mesenchymal transition (EMT) in MDCK cells. The promotion of EMT was dependent on podoplanin binding to ERM (ezrin, radixin, moesin) proteins through its cytoplasmic (CT) domain, which led to RhoA-associated kinase (ROCK)-dependent ERM phosphorylation. Using detergent-resistant membrane (DRM) assays, as well as transmembrane (TM) interactions and ganglioside GM1 binding, we present evidence supporting the localization of podoplanin in raft platforms important for cell signalling. Podoplanin mutant constructs harbouring a heterologous TM region or lacking the CT tail were unable to associate with DRMs, stimulate ERM phosphorylation and promote EMT or cell migration. Similar effects were observed upon disruption of a GXXXG motif within the TM domain, which is involved in podoplanin self-assembly. In contrast, deletion of the extracellular (EC) domain did not affect podoplanin DRM association. Together, these data suggest that both the CT and TM domains are required for podoplanin localization in raft platforms, and that this association appears to be necessary for podoplanin-mediated EMT and cell migration.     

Retention of a recombinant GFP protein expressed by the yellow fever 17D virus in the E/NS1 intergenic region in the endoplasmic reticulum

The flaviviral envelope proteins, E protein and precursor membrane protein, are mainly associated with the endoplasmic reticulum (ER) through two transmembrane (TM) domains that are exposed to the luminal face of this compartment. Their retention is associated with the viral assembly process. ER-retrieval motifs were mapped at the carboxy terminus of these envelope proteins. A recombinant yellow fever (YF) 17D virus expressing the reporter green fluorescent protein (GFP) with the stem-anchor (SA) region of E protein fused to its carboxy terminus was subjected to distinct genetic mutations in the SA sequence to investigate their effect on ER retention. Initially, we introduced progressive deletions of the stem elements (H1, CS and H2). In a second set of mutants, the effect of a length increase for the first TM anchor region was evaluated either by replacing it with the longer TM of human LAMP-1 or by the insertion of the VALLLVA sequence into its carboxy terminus. We did not detect any effect on the GFP localisation in the cell, which remained associated with the ER. Further studies should be undertaken to elucidate the causes of the ER retention of recombinant proteins expressed at the intergenic E/NS1 region of the YF 17D virus polyprotein.     

Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell.     

Functional ATPase activity of p97/valosin-containing protein (VCP) is required for the quality control of endoplasmic reticulum in neuronally differentiated mammalian PC12 cells

Abnormal protein accumulation and cell death with cytoplasmic vacuoles are hallmarks of several neurodegenerative disorders. We previously identified p97/valosin-containing protein (VCP), an AAA ATPase with two conserved ATPase domains (D1 and D2), as an interacting partner of the Machado-Joseph disease (MJD) protein with expanded polyglutamines that causes Machado-Joseph disease. To reveal its pathophysiological roles in neuronal cells, we focused on its ATPase activity. We constructed and characterized PC12 cells expressing wild-type p97/VCP and p97(K524A), a D2 domain mutant. The expression level, localization, and complex formation of both proteins were indistinguishable, but the ATPase activity of p97(K524A) was much lower than that of the wild type. p97(K524A) induced cytoplasmic vacuoles that stained with an endoplasmic reticulum (ER) marker, and accumulation of polyubiquitinated proteins in the nuclear and membrane but not cytoplasmic fractions was observed, together with the elevation of ER stress markers. These results show that p97/VCP is essential for degrading membrane-associated ubiquitinated proteins and that profound deficits in its ATPase activity severely affect ER quality control, leading to abnormal ER expansion and cell death. Excessive accumulation of misfolded proteins may inactivate p97/VCP in several neurodegenerative disorders, eventually leading to the neurodegenerations.     

The retrieval function of the KDEL receptor requires PKA phosphorylation of its C-terminus

The KDEL receptor is a Golgi/intermediate compartment-located integral membrane protein that carries out the retrieval of escaped ER proteins bearing a C-terminal KDEL sequence. This occurs throughout retrograde traffic mediated by COPI-coated transport carriers. The role of the C-terminal cytoplasmic domain of the KDEL receptor in this process has been investigated. Deletion of this domain did not affect receptor subcellular localization although cells expressing this truncated form of the receptor failed to retain KDEL ligands intracellularly. Permeabilized cells incubated with ATP and GTP exhibited tubular processes-mediated redistribution from the Golgi area to the ER of the wild-type receptor, whereas the truncated form lacking the C-terminal domain remained concentrated in the Golgi. As revealed with a peptide-binding assay, this domain did not interact with both coatomer and ARF-GAP unless serine 209 was mutated to aspartic acid. In contrast, alanine replacement of serine 209 inhibited coatomer/ARF-GAP recruitment, receptor redistribution into the ER, and intracellular retention of KDEL ligands. Serine 209 was phosphorylated by both cytosolic and recombinant protein kinase A (PKA) catalytic subunit. Inhibition of endogenous PKA activity with H89 blocked Golgi-ER transport of the native receptor but did not affect redistribution to the ER of a mutated form bearing aspartic acid at position 209. We conclude that PKA phosphorylation of serine 209 is required for the retrograde transport of the KDEL receptor from the Golgi complex to the ER from which the retrieval of proteins bearing the KDEL signal depends.     

Deletion of the cytoplasmic domain of human alpha3/4 fucosyltransferase III causes the shift of the enzyme to early Golgi compartments

The transmembrane domain (TM) and flanking regions of glycosyltransferases (GTs) have been implicated in the localization of these proteins in the Golgi apparatus (GA). alpha3/4 Fucosyltransferase III (FT3wt) (EC 2.4.1.65) is localized in the trans-Golgi and trans-Golgi network (TGN) of baby hamster kidney (BHK) cells and synthesizes Lewis determinants associated with cell adhesion events. We have evaluated the effect of removing the cytosolic domain on the localization of the enzyme and its capacity for synthesizing the Lewis A (Le A) determinant. The mutant where the cytoplasmic domain (Asp-2 to Trp-13) of FT3wt has been deleted (FT3dc) was localized in the Golgi but it was shifted to earlier compartments than FT3wt. The mutant was not detected on the plasma membrane (PM) and glycosylation analysis indicated that FT3dc was transported beyond the endoplasmic reticulum (ER) since complex type glycosylation was observed. Cells expressing FT3dc showed a significantly lower efficiency to synthesize Le A when compared with cells expressing FT3wt, in vivo. This reduction was not due to lower specific activity because both enzyme forms had a similar specific activity in vitro. Therefore, removal of FT3 cytosolic tail caused a shift in enzyme distribution to earlier Golgi compartments concomitant to the decrease of its biosynthetic capacity.     

Modeling the Tertiary Structure of the Patatin Domain of Neuropathy Target Esterase

Neuropathy target esterase (NTE) is a transmembrane protein of unknown function whose specific chemical modification by certain organophosphorus (OP) compounds leads to distal axonopathy. Therefore, solving the 3D structure of NTE would advance the understanding of its pathogenic and physiologic roles. In this study, the tertiary structures of the patatin (catalytic) domain and the N-terminal transmembrane domain of NTE were modeled using the crystal structures of patatin (PDB ID 1oxw) and moricin (PDB ID 1kv4) as templates. Sequence alignments and secondary structure predictions were obtained from the INUB server (Buffalo, NY). O and PyMol were used to build the PNTE and NTE TMD chains from these sequence alignments. The PNTE model was refined in the presence of water using the crystallography and NMR system, while the NTE TMD model was refined in vacuo using the GROMOS implementation in the Swiss PDB viewer. The modeled active site of NTE was found to consist of a Ser966-Asp1086 catalytic dyad, which is characteristic of phospholipase A2 enzymes. The Ser966 Ogamma was located 2.93 A from the Odelta2 of Asp1086. In addition, our NTE model was found to contain a single N-terminal transmembrane domain. This modeling effort provided structural and mechanistic predictions about the catalytic domain of NTE that are being verified via experimental techniques.     

Roles of multimerization and membrane association in the proteolytic functions of FtsH (HflB)

FtsH (HflB) is an Escherichia coli ATP-dependent protease that degrades some integral membrane and cytoplasmic proteins. While anchored to the cytoplasmic membrane by the two transmembrane (TM) segments near the N-terminus, it has a large cytoplasmic domain. The N-terminal region also has a role in homo-oligomerization of this protein. To study the significance of the membrane integration and oligomer formation, we constructed FtsH derivatives in which the N-terminal region had been deleted or replaced with either the leucine zipper sequence from Saccharomyces cerevisiae GCN4 protein or TM regions from other membrane proteins. The cytoplasmic domain, which was monomeric and virtually inactive, was converted, by the attachment of the leucine zipper, to an oligomer with proteolytic function against a soluble, but not a membrane-bound substrate. In contrast, chimeric TM-FtsH proteins were active against both substrate classes. We suggest that the cytoplasmic domain has intrinsic but weak self-interaction ability, which becomes effective with the aid of the leucine zipper or membrane tethering, and that membrane association is essential for FtsH to degrade integral membrane proteins.