Identification of a chemoreceptor zinc-binding domain common to cytoplasmic bacterial chemoreceptors

We report the identification and characterization of a previously unidentified protein domain found in bacterial chemoreceptors and other bacterial signal transduction proteins. This domain contains a motif of three noncontiguous histidines and one cysteine, arranged as Hxx[WFYL]x(21-28)Cx[LFMVI]Gx[WFLVI]x(18-27)HxxxH(boldface type indicates residues that are nearly 100% conserved). This domain was first identified in the soluble Helicobacter pylori chemoreceptor TlpD. Using inductively coupled plasma mass spectrometry on heterologously and natively expressed TlpD, we determined that this domain binds zinc with a subfemtomolar dissociation constant. We thus named the domain CZB, for chemoreceptor zinc binding. Further analysis showed that many bacterial signaling proteins contain the CZB domain, most commonly proteins that participate in chemotaxis but also those that participate in c-di-GMP signaling and nitrate/nitrite sensing, among others. Proteins bearing the CZB domain are found in several bacterial phyla. The variety of signaling proteins using the CZB domain suggests that it plays a critical role in several signal transduction pathways.     

The highly conserved domain of the Caulobacter McpA chemoreceptor is required for its polar localization

We have fused GFP to the C-terminus of McpA to study chemoreceptor polar localization in Caulobacter crescentus. The full-length McpA-GFP fusion is polarly localized and methylated. The methylation is dependent on the chemoreceptor methyltransferase (cheR) and chemoreceptor methylesterase (cheB) genes present in the mcpA operon. C-terminal and internal deletions of McpA were constructed and fused to the N-terminus of GFP to identify the domains required for polar localization. When the R1 methylation domain was deleted, the McpA-GFP fusion was still polarly localized, suggesting that this domain is dispensable for polar localization. However, when the highly conserved domain (HCD), which is involved in interacting with CheW, was deleted either by an internal deletion or C-terminal deletion, the resulting McpA-GFP fusions were completely delocalized. When the mcpA operon, which contains the cheW and cheA homologues, was deleted, the full-length McpA-GFP fusion was delocalized. Although additional chemotaxis genes are required for the polar localization of McpA-GFP, the presence of the single polar flagellum is not required. However, in filamentous cells, which are frequently found in C. crescentus fliF mutants, the McpA-GFP fusion was observed at mid-cell positions.     

Degradation of a Caulobacter soluble cytoplasmic chemoreceptor is ClpX dependent

In order to determine whether ClpXP-mediated proteolysis is a common mechanism used to regulate the chemotaxis machinery during the cell cycle of Caulobacter crescentus, we have characterized a soluble cytoplasmic chemoreceptor, McpB. The mcpB gene lies adjacent to the major chemotaxis operon, which encodes 12 chemotaxis proteins, including the membrane chemoreceptor McpA. Like McpA, McpB possesses a C-terminal CheBR docking motif and three potential methylation sites, which we suggest are methylated. The McpB protein is degraded via a ClpX-dependent pathway during the swarmer-to-stalked cell transition, and a motif, which is 3 amino acids N-terminal to the McpB CheBR docking site, is required for proteolysis. Analysis of the degradation signal in McpB and McpA reveals a common motif present in the other four chemoreceptors that possess CheBR docking sites. A green fluorescent protein (GFP) fusion bearing 58 amino acids from the C terminus of McpA, which contains this motif, is degraded, suggesting that the C-terminal sequence is sufficient to confer ClpXP protease susceptibility.     

Shortened cytoplasmic domain affects intracellular transport but not nuclear localization of a viral glycoprotein

Herpes simplex virus (HSV) buds from the inner nuclear membrane of the infected cells. The glycoprotein gB-1 of HSV contains a stretch of 69 hydrophobic amino acids near the COOH terminus and a 109-amino acid cytoplasmic domain. By oligonucleotide-directed mutagenesis, five gB-1 mutants were constructed which either lack a cytoplasmic tail or contained 3, 6, 22, or 43 amino acids in the cytoplasmic tail. When expressed in COS cells all of the mutant glycoproteins were synthesized but the rate of intracellular transport and the appearance at the cell surface of the mutant gB-1 protein lacking the cytoplasmic tail or containing 3 and 6 amino acids in the cytoplasmic domain was drastically reduced. The wild-type gB-1 as well as all of the mutants in the cytoplasmic tail were, however, located on the nuclear envelope. These results suggest that the cytoplasmic domain of the glycoprotein gB may play a role in intracellular transport but not in the nuclear localization.     

An essential cytoplasmic domain for the Golgi localization of coiled-coil proteins with a COOH-terminal membrane anchor

Giantin is a resident Golgi protein that has an extremely long cytoplasmic domain (about 370 kDa) and is anchored to the Golgi membrane by the COOH-terminal membrane-anchoring domain (CMD) with no luminal extension. We examined the essential domain of giantin required for Golgi localization by mutational analysis. The Golgi localization of giantin was not affected by the deletion of its CMD or by substitution with the CMD of syntaxin-2, a plasma membrane protein. The giantin CMD fused to the cytoplasmic domain of syntaxin-2 could not retain the chimera in the Golgi apparatus. Sequential deletion analysis showed that the COOH-terminal sequence (positions 3059--3161) adjacent to the CMD was the essential domain required for the Golgi localization of giantin. We also examined two other Golgi-resident proteins, golgin-84 and syntaxin-5, with a similar membrane topology as giantin. It was confirmed that the cytoplasmic domain of about 100 residues adjacent to the CMD was required for their Golgi localization. Taken together, these results suggest that the COOH-terminally anchored Golgi proteins with long cytoplasmic extensions have the Golgi localization signal(s) in the cytoplasmic sequence adjacent to the CMD. This is in contrast to previous observations that a transmembrane domain is required for Golgi localization by other Golgi proteins transported from the endoplasmic reticulum.     

Ligand-dependent and -independent integrin focal contact localization: the role of the alpha chain cytoplasmic domain.

Many integrin receptors localize to focal contact sites upon binding their ligand. However, unoccupied integrin receptors do not localize to focal contact sites. Because the integrin beta 1 cytoplasmic domain appears to have a focal contact localization signal, there must be a mechanism by which this domain is kept inactive in the unoccupied state and becomes exposed or activated in the occupied receptor. We considered that this mechanism involves the alpha subunit cytoplasmic domain. To test this hypothesis, we have established two NIH 3T3 cell lines that express either the human alpha 1 wild-type subunit (HA1 cells) or the cytoplasmic domain deleted alpha 1 subunit (CYT cells). Both cell lines express similar levels of the human alpha 1 subunit, and there is no significant effect of the deletion on the dimerization and surface expression of the receptor. Furthermore, the deletion had no effect on the binding or adhesion via alpha 1 beta 1 to its ligand collagen IV. However, when these two cell lines are plated on fibronectin (FN), which is a ligand for alpha 5 beta 1 but not for alpha 1 beta 1, there is a striking difference in the cellular localization of alpha 1 beta 1. The HA1 cells show only alpha 5 in focal contacts, without alpha 1, demonstrating that all of the integrin localization is ligand dependent. In contrast, when the CYT cells are plated on FN, the mutant alpha 1 appears in focal contacts along with the alpha 5/beta 1. Thus, there is both ligand-dependent (alpha 5/beta 1) and ligand-independent (alpha 1/beta 1) focal contact localization in these cells. The truncated alpha 1 also localized to focal contacts in a ligand-independent manner on vitronectin. We conclude that the mutant alpha 1 no longer requires ligand occupancy for focal contact localization. These data strongly suggest that the alpha cytoplasmic domain plays a role in the normal ligand-dependent integrin focal contact localization.     

Stabilization of polar localization of a chemoreceptor via its covalent modifications and its communication with a different chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.     

Implementation of a Hebbian chemoreceptor model for diffusive source localization

While new approaches to chemical localization have been proposed, animals are still widely used for locating landmines and illegal substances. Existing electronic noses still do not have the necessary sensitivity and accuracy. By modeling a cell’s chemical detection system, we can gain insight into the basic “olfactory” system. We use an inspiration from chemotaxis and Hebbian learning to enhance localization and tracking of gradient sources, which can be applied to both chemicals and heat. The eukaryotic receptor clustering model shows improvement over previous prokaryotic chemotaxis-inspired methods that do not take into account receptor clustering. Receptor clustering essentially adapts receptors spatio-temporally. For a mobile simulation, our method locates the source in less convergence time than the other chemotaxis algorithms and insignificantly less time compared to no spatio-temporal filtering (e.g. a single-sensor memoryless case). We then show that local regions of receptor cooperation have the best performance reflecting observations of receptor behavior in biology. To demonstrate the performance of this system in real-time, a stationary 4/8-sensor version of the array is implemented, and the algorithm improves the convergence time, mean, and variance of the Direction-of-Arrival calculation in diffusive, turbulent, and noisy environments.     

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.     

Secondary structure of a KCNE cytoplasmic domain

Type I transmembrane KCNE peptides contain a conserved C-terminal cytoplasmic domain that abuts the transmembrane segment. In KCNE1, this region is required for modulation of KCNQ1 K(+) channels to afford the slowly activating cardiac I(Ks) current. We utilized alanine/leucine scanning to determine whether this region possesses any secondary structure and to identify the KCNE1 residues that face the KCNQ1 channel complex. Helical periodicity analysis of the mutation-induced perturbations in voltage activation and deactivation kinetics of KCNQ1-KCNE1 complexes defined that the KCNE1 C terminus is alpha-helical when split in half at a conserved proline residue. This helical rendering assigns all known long QT mutations in the KCNE1 C-terminal domain as protein facing. The identification of a secondary structure within the KCNE1 C-terminal domain provides a structural scaffold to map protein-protein interactions with the pore-forming KCNQ1 subunit as well as the cytoplasmic regulatory proteins anchored to KCNQ1-KCNE complexes.     

Functionally essential cytoplasmic domain of the erythropoietin receptor.

The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.     

Fc receptor isoforms exhibit distinct abilities for coated pit localization as a result of cytoplasmic domain heterogeneity

Mouse macrophages and lymphocytes express two distinct isoforms of a single class of Fc receptor for IgG. The macrophage isoform (FcRII-B2) is identical to the lymphocyte isoform (FcRII-B1) except for an inframe insertion in the cytoplasmic tail of FcRII-B1 that increases its length from 47 to 94 amino acids. To determine the functional significance of this cytoplasmic domain variation, presumably the result of alternative mRNA splicing, we expressed both isoforms in receptor-negative fibroblasts. While FcRII-B2 mediated the efficient ligand internalization and delivery to lysosomes, endocytosis via FcRII-B1--and via a tailminus mutant--was relatively inefficient. This difference reflected the inability of FcRII-B1 (and the tailminus mutant) to accumulate in clathrin-coated pits. Thus, the FcRII-B2 cytoplasmic tail contains a domain needed for accumulation in coated pits, and this domain is disrupted by the 47 amino acid insertion in FcRII-B1.     

An acidic sequence within the cytoplasmic domain of furin functions as a determinant of trans-Golgi network localization and internalization from the cell surface.

The mammalian endopeptidase, furin, is predominantly localized to the trans-Golgi network (TGN) at steady state. The localization of furin to this compartment seems to be the result of a dynamic process in which the protein undergoes cycling between the TGN and the plasma membrane. Both TGN localization and internalization from the plasma membrane are mediated by targeting information contained within the cytoplasmic domain of furin. Here, we report the results of a mutagenesis analysis aimed at identifying the source(s) of targeting information within the furin cytoplasmic domain. Our studies show that there are at least two cytoplasmic determinants that contribute to the steady-state localization and trafficking of furin. The first determinant corresponds to a canonical tyrosine-based motif, YKGL (residues 758-761), that functions mainly as an internalization signal. The second determinant consists of a strongly hydrophilic sequence (residues 766-783) that contains a large cluster of acidic residues (E and D) and is devoid of any tyrosine-based or di-leucine-based motifs. This second determinant is capable of conferring localization to the TGN as well as mediating internalization from the plasma membrane. Thus, these observations establish the existence of a novel, autonomous determinant distinct from sorting signals described previously.     

Nucleolar and cytoplasmic localization of annexin V.

The subcellular localization of annexin V in cultured human umbilical vein endothelial cells, epithelial cells and fibroblasts was examined. Indirect immunofluorescence and immunoblotting studies using affinity-purified anti-annexin V antibodies revealed that annexin V is located within the cytoplasm and nucleus of these cells. Further examination and direct binding studies showed that annexin V within the nucleus is associated with the nucleolus. These findings suggest that annexin V may play a role in a nucleolar function, such as ribosome assembly and transport.     

Two independent targeting signals in the cytoplasmic domain determine trans-Golgi network localization and endosomal trafficking of the proprotein convertase furin.

Furin, a subtilisin-like eukaryotic endoprotease, is responsible for proteolytic cleavage of cellular and viral proteins transported via the constitutive secretory pathway. Cleavage occurs at the C-terminus of basic amino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golgi network (TGN), but also in clathrin-coated vesicles dispatched from the TGN, on the plasma membrane as an integral membrane protein and in the medium as an anchorless enzyme. When furin was vectorially expressed in normal rat kidney (NRK) cells it accumulated in the TGN similarly to the endogenous glycoprotein TGN38, often used as a TGN marker protein. The signals determining TGN targeting of furin were investigated by mutational analysis of the cytoplasmic tail of furin and by using the hemagglutinin (HA) of fowl plague virus, a protein with cell surface destination, as a reporter molecule, in which membrane anchor and cytoplasmic tail were replaced by the respective domains of furin. The membrane-spanning domain of furin grafted to HA does not localize the chimeric molecule to the TGN, whereas the cytoplasmic domain does. Results obtained on furin mutants with substitutions and deletions of amino acids in the cytoplasmic tail indicate that wild-type furin is concentrated in the TGN by a mechanism involving two independent targeting signals, which consist of the acidic peptide CPSDSEEDEG783 and the tetrapeptide YKGL765. The acidic signal in the cytoplasmic domain of a HA-furin chimera is necessary and sufficient to localize the reporter molecule to the TGN, whereas YKGL is a determinant for targeting to the endosomes. The data support the concept that the acidic signal, which is the dominant one, retains furin in the TGN, whereas the YKGL motif acts as a retrieval signal for furin that has escaped to the cell surface.     

Human fucosyltransferase IX: specificity towards N-linked glycoproteins and relevance of the cytoplasmic domain in intra-Golgi localization

The alpha3-fucosyltransferase IX (FUT9) catalyses the transfer of fucose in an alpha3 linkage onto terminal type II (Galbeta4GlcNAc) acceptors, the final step in the biosynthesis of the Lewis(x) (Le(x)) epitope, in neurons. In this work, FUT9 cloned from NT2N neurons and overexpressed in HeLa cells (FUT9wt), was found to efficiently fucosylate asialoerythropoietin (asialoEPO), and bovine asialofetuin, but not sialylated EPO. Analysis by HPAEC-PAD and MALDI/TOF-MS revealed predominantly mono-fucosylation by FUT9wt of type II di-, tri- and tetraantennary N-glycans with proximal fucose, with and without N-acetylactosamine repeats from asialoEPO. Minor amounts of difucosylated structures were also found. The results suggested that FUT9 could fucosylate Le(x) carrier-glycoproteins in neurons. Furthermore, FUT9wt was found to be activated by Mn(2+) and it was capable of synthesizing Le(a), although to a lesser extent than Le(x) and Le(y). In vivo, HeLa cells transfected with FUT9wt expressed de novo Le(x), as detected by immunofluorescence microscopy. FUT9 was found to be a trans-Golgi and trans-Golgi network (TGN) glycosyltransferase from confocal immunofluorescence co-localization with the markers of the secretory pathway beta4-galactosyltransferase (trans-Golgi and TGN) and TGN-46 (TGN). Deletion of the cytoplasmic domain caused a shift to the cis-Golgi, thus suggesting that information for intra-Golgi localization is contained within the cytoplasmic domain.