Topogenesis of membrane proteins: determinants and dynamics.

For targeting and integration of proteins into the mammalian endoplasmic reticulum, two types of signals can be distinguished: those that translocate their C-terminal sequence (cleavable signals and signal-anchors) and those that translocate their N-terminus (reverse signal-anchors). In addition to the well established effect of flanking charges, also the length and hydrophobicity of the apolar core of the signal as well as protein folding and glycosylation contribute to orienting the signal in the translocon. In multi-spanning membrane proteins, topogenic determinants are distributed throughout the sequence and may even compete with each other. During topogenesis, segments of up to 60 residues may move back and forth through the translocon, emphasizing unexpected dynamic aspects of topogenesis.

Evaluation of a pseudo-R2 measure for panel probit models.

A simulation study designed to evaluate the pseudo-R2T proposed in an earlier paper by Spiess and Keller suggests that, for the models considered, this measure represents the goodness of fit not only of the systematic part, but also of the assumed correlation structure in binary panel probit models.

In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation.

We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [(32)P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be approximately 1.6 times the rate of translation.

A novel assay to demonstrate an intersection of the exocytic and endocytic pathways at early endosomes.

The mechanism of transport of membrane proteins from the trans-Golgi to the cell surface is still poorly understood. Previous studies suggested that basolateral membrane proteins, such as the transferrin receptor and the asialoglycoprotein receptor H1, take an indirect route to the plasma membrane via an intracellular, most likely endosomal intermediate. To define this compartment we developed a biochemical assay based on the very definition of endosomes. The assay is based on internalizing anti-H1 antibodies via the endocytic cycle of the receptor itself. Internalized antibody formed immune complexes with newly synthesized H1, which had been pulse-labeled with [(35)S]sulfate and chased out of the trans-Golgi for a period of time that was insufficient for H1 to reach the surface. Hence, antibody capture occurred intracellularly. Double-immunofluorescence labeling demonstrated that antibody-containing compartments also contained transferrin and thus corresponded to early and recycling endosomes. The results therefore demonstrate an intracellular intersection of the exocytic and endocytic pathways with implications for basolateral sorting.

Crystal structure of the carbohydrate recognition domain of the H1 subunit of the asialoglycoprotein receptor.

The human asialoglycoprotein receptor (ASGPR), also called hepatic lectin, is an integral membrane protein and is responsible for the clearance of desialylated, galactose-terminal glycoproteins from the circulation by receptor-mediated endocytosis. It can be subdivided into four functional domains: the cytosolic domain, the transmembrane domain, the stalk and the carbohydrate recognition domain (CRD). The galactose-binding domains belong to the superfamily of C-type (calcium-dependent) lectins, in particular to the long-form subfamily with three conserved intramolecular disulphide bonds. It is able to bind terminal non-reducing galactose residues and N-acetyl-galactosamine residues of desialated tri or tetra-antennary N-linked glycans. The ASGPR is a potential liver-specific receptor for hepatitis B virus and Marburg virus and has been used to target exogenous molecules specifically to hepatocytes for diagnostic and therapeutic purposes.Here, we present the X-ray crystal structure of the carbohydrate recognition domain of the major subunit H1 at 2.3 A resolution. While the overall fold of this and other known C-type lectin structures are well conserved, the positions of the bound calcium ions are not, indicating that the fold is stabilised by alternative mechanisms in different branches of the C-type lectin family. It is the first CRD structure where three calcium ions form an intergral part of the structure. In addition, the structure provides direct confirmation for the conversion of the ligand-binding site of the mannose-binding protein to an asialoglycoprotein receptor-like specificity suggested by Drickamer and colleagues. In agreement with the prediction that the coiled-coil domain of the ASGPR is separated from the CRD and its N-terminal disulphide bridge by several residues, these residues are indeed not alpha-helical, while in tetranectin they form an alpha-helical coiled-coil.

The topogenic contribution of uncharged amino acids on signal sequence orientation in the endoplasmic reticulum.

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C- or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val(19), Trp(19), and Tyr(22) generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a position-dependent effect, which raises the possibility of a conformational contribution to topogenesis.

Basolateral sorting signals differ in their ability to redirect apical proteins to the basolateral cell surface.

Polarized sorting of membrane proteins in epithelial cells is mediated by cytoplasmic basolateral signals or by apical signals in the transmembrane or exoplasmic domains. Basolateral signals were generally found to be dominant over apical determinants. We have generated chimeric proteins with the cytoplasmic domain of either the asialoglycoprotein receptor H1 or the transferrin receptor, two basolateral proteins, fused to the transmembrane and exoplasmic segments of aminopeptidase N, an apical protein, and analyzed them in Madin-Darby canine kidney cells. Whereas both cytoplasmic sequences induced endocytosis of the chimeras, only that of the transferrin receptor mediated basolateral expression in steady state. The H1 fusion protein, although still largely sorted to the basolateral side in biosynthetic surface transport, was subsequently resorted to the apical cell surface. We tested whether the difference in sorting between trimeric wild-type H1 and the dimeric aminopeptidase chimera was caused by the number of sorting signals presented in the oligomers. Consistent with this hypothesis, the H1 signal was fully functional in a tetrameric fusion protein with the transmembrane and exoplasmic domains of influenza neuraminidase. The results suggest that basolateral signals per se need not be dominant over apical determinants for steady-state polarity and emphasize an important contribution of the valence of signals in polarized sorting.

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis.

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.

Glycosylation can influence topogenesis of membrane proteins and reveals dynamic reorientation of nascent polypeptides within the translocon.

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH(2)-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH(2)-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.

Recognition of sorting signals by clathrin adaptors.

Sorting of membrane proteins is generally mediated by cytosolic coats, which create a scaffold to form coated buds and vesicles and to selectively concentrate cargo by interacting with cytosolic signals. The classical paradigm is the interaction between clathrin coats and associated adaptor proteins, which cluster receptors with characteristic tyrosine and dileucine motifs during endocytosis. Clathrin in association with different sets of adaptors is found in addition at the trans-Golgi network and endosomes. Sequences similar to internalization signals also direct lysosomal and basolateral sorting, which implicates related clathrinadaptor coats in the respective sorting pathways. This review concentrates on the recognition of sorting signals by clathrin-associated adaptor proteins, an area of significant recent progress due to new methodological and conceptual approaches.

Isolation, electron microscopic imaging, and 3-D visualization of native cardiac thin myofilaments.

An increasing number of cardiac diseases are currently pinpointed to reside at the level of the thin myofilaments (e.g., cardiomyopathies, reperfusion injury). Hence the aim of our study was to develop a new method for the isolation of mammalian thin myofilaments suitable for subsequent high-resolution electron microscopic imaging. Native cardiac thin myofilaments were extracted from glycerinated porcine myocardial tissue in the presence of protease inhibitors. Separation of thick and thin myofilaments was achieved by addition of ATP and several centrifugation steps. Negative staining and subsequent conventional and scanning transmission electron microscopy (STEM) of thin myofilaments permitted visualization of molecular details; unlike conventional preparations of thin myofilaments, our method reveals the F-actin moiety and allows direct recognition of thin myofilament-associated porcine cardiac troponin complexes. They appear as "bulges" at regular intervals of approximately 36 nm along the actin filaments. Protein analysis using SDS-polyacrylamide gel electrophoresis revealed that only approximately 20% troponin I was lost during the isolation procedure. In a further step, 3-D helical reconstructions were calculated using STEM dark-field images. These 3-D reconstructions will allow further characterization of molecular details, and they will be useful for directly visualizing molecular alterations related to diseased cardiac thin myofilaments (e.g., reperfusion injury, alterations of Ca2+-mediated tropomyosin switch).

Mechanism of endoplasmic reticulum retention of mutant vasopressin precursor caused by a signal peptide truncation associated with diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus is caused by mutations in the gene encoding the vasopressin precursor protein, prepro-vasopressin-neurophysin II. We analyzed the molecular consequences of a mutation (DeltaG227) recently identified in a Swiss kindred that destroys the translation initiation codon. In COS-7 cells transfected with the mutant cDNA, translation was found to initiate at an alternative ATG, producing a truncated signal sequence that was functional for targeting and translocation but was not cleaved by signal peptidase. The mutant precursor was completely retained within the endoplasmic reticulum. The uncleaved signal did not affect folding of the neurophysin portion of the precursor, as determined by its protease resistance. However, formation of disulfide-linked aggregates indicated that it interfered with the formation of the disulfide bond in vasopressin, most likely by blocking its insertion into the hormone binding site of neurophysin. Preventing disulfide formation in the vasopressin nonapeptide by mutation of cysteine 6 to serine was shown to be sufficient to cause aggregation and retention. These results indicate that the DeltaG227 mutation induces translation of a truncated signal sequence that cannot be cleaved but prevents correct folding and oxidation of vasopressin, thereby causing precursor aggregation and retention in the endoplasmic reticulum.

A mixed approach and a distribution-free multiple imputation technique for the estimation of a multivariate probit model with missing values.

In the present paper a mixed generalized estimating/pseudo-score equations (GEPSE) approach together with a distribution-free multiple imputation technique is proposed for the estimation of regression and correlation structure parameters of multivariate probit models with missing values for an ordered categorical time-invariant variable. Furthermore, a generalization of the squared trace correlation (RT2) for multivariate probit models, denoted by pseudo-RT2, is proposed. A simulation study was conducted, simulating a probit model with an equicorrelation structure in the errors of an underlying regression model and using two different missing mechanisms. For a low 'true' correlation the difference between the GEPSE, a generalized estimating equations (GEE) and a maximum likelihood (ML) estimator were negligible. For a high 'true' correlation the GEPSE estimator turned out to be more efficient than the GEE and very efficient relative to the ML estimator. Furthermore, the pseudo-RT2 was close to RT2 of the underlying linear model. The mixed approach is illustrated using a psychiatric data set of depressive in-patients. The results of this analysis suggest that the depression score at discharge from a psychiatric hospital and the occurrence of stressful life events seem to increase the probability of having an episode of major depression within a one-year interval after discharge. Furthermore, the correlation structure points to short-time effects on having or not having a depressive episode, not accounted for in the systematic part of the regression model.

Ligand-induced endocytosis of the asialoglycoprotein receptor: evidence for heterogeneity in subunit oligomerization.

The hepatic asialoglycoprotein receptor, a noncovalent hetero-oligomer of two subunits, is a constitutively cycling endocytic receptor. However, the ligand asialoorosomucoid caused downregulation of up to 40% of surface binding sites and a twofold increase in internalization rate. This was not the result of receptor crosslinking, since monovalent ligands had the same effect. Ligand binding thus appears to transmit a signal to the cytosolic portion of the receptor not unlike in signaling receptors. The two subunits were endocytosed at different average rates lower than that of ligand, indicating heterogeneity in oligomer formation and potentially in ligand specificity.

Distinct subcellular localization of calcium binding S100 proteins in human smooth muscle cells and their relocation in response to rises in intracellular calcium.

Changes in cytosolic Ca2+ concentration control a wide range of cellular responses, and intracellular Ca2+-binding proteins are the key molecules to transduce Ca2+ signaling via interactions with different types of target proteins. Among these, S100 Ca2+-binding proteins, characterized by a common structural motif, the EF-hand, have recently attracted major interest due to their cell- and tissue-specific expression pattern and involvement in various pathological processes. The aim of our study was to identify the subcellular localization of S100 proteins in vascular smooth muscle cell lines derived from human aorta and intestinal smooth muscles, and in primary cell cultures derived from arterial smooth muscle tissue under normal conditions and after stimulation of the intracellular Ca2+ concentration. Confocal laser scanning microscopy was used with a specially designed colocalization software. Distinct intracellular localization of S100 proteins was observed: S100A6 was present in the sarcoplasmic reticulum as well as in the cell nucleus. S100A1 and S100A4 were found predominantly in the cytosol where they were strongly associated with the sarcoplasmic reticulum and with actin stress fibers. In contrast, S100A2 was located primarily in the cell nucleus. Using a sedimentation assay and subsequent electron microscopy after negative staining, we demonstrated that S100A1 directly interacts with filamentous actin in a Ca2+-dependent manner. After thapsigargin (1 microM) induced increase of the intracellular Ca2+ concentration, specific vesicular structures in the sarcoplasmic reticulum region of the cell were formed with high S100 protein content. In conclusion, we demonstrated a distinct subcellular localization pattern of S100 proteins and their interaction with actin filaments and the sarcoplasmic reticulum in human smooth muscle cells. The specific translocation of S100 proteins after intracellular Ca2+ increase supports the hypothesis that S100 proteins exert several important functions in the regulation of Ca2+ homeostasis in smooth muscle cells.

The role of the hydrophobic domain in orienting natural signal sequences within the ER membrane.

The orientation of signal sequences during insertion into the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar domain. Using recombinant and mutant proteins, also length and hydrophobicity of the apolar segment were shown to affect the orientation: translocation of the N-terminus was found to be favored by long hydrophobic sequences, and translocation of the C-terminus, by short ones. Here, we tested the physiological significance of this phenomenon by mutagenesis of the hydrophobic portion of two natural signals with unusual flanking charges. Extending the hydrophobic domain of the short, cleaved Ncyt/Cexo signal of pre-provasopressin-neurophysin II and shortening that of the Nexo/Ccyt signal anchor of microsomal epoxide hydrolase resulted in a significant fraction of polypeptides inserting in the opposite orientation to that of the wild-type proteins. The topogenic contribution of the hydrophobic domain is thus important for the correct and uniform orientation of natural proteins and can explain the behavior of some of the signals with unusual flanking charges.

Experimental technique for (laparoscopic) bowel anastomosis: transient endoluminally stented anastomosis (TESA).

A new technique for bowel anastomosis is presented. The principle of transient endoluminally stented anastomosis (TESA) is based on anastomosing the two bowel ends around a resorbable stent of polyglycolic acid (PGA) in seroserosal contact. To evaluate the feasibility of TESA for bowel anastomosis, laparoscopic colon anastomosis following sigma resection was performed in five juvenile pigs. Three animals were sacrificed 2 months postoperatively, and the anastomoses were examined radiologically and histologically. One animal was sacrificed at day 2, suffering from acute peritonitis due to small bowel leak but with regular colon anastomosis. One trial was terminated at the fourth postoperative day because of insufficiency of the colon anastomosis. Three animals did not have any complications during the 2-month follow-up. In these animals the colon anastomoses were not detectable radiologically at the time of death. The microscopic examination showed intact mucosal and muscular layers without foreign material. Our study demonstrates that laparoscopic application of TESA to colon anastomosis is a feasible method. These results will further stimulate our future research for an anastomosis technique avoiding remnant foreign material.

Multiple determinants direct the orientation of signal-anchor proteins: the topogenic role of the hydrophobic signal domain.

The orientation of signal-anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7-25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.

The oligomerization domain of the asialoglycoprotein receptor preferentially forms 2:2 heterotetramers in vitro.

The human hepatic asialoglycoprotein receptor is a noncovalent hetero-oligomer composed of two homologous subunits, H1 and H2, with an as yet unknown stoichiometry. Ligand specificity and binding affinity depend on the arrangement of the subunits in the complex. An 80-amino acid segment connecting the transmembrane and the carbohydrate binding domains contains heptad repeats characteristic of alpha-helical coiled coil structure. We expressed and purified corresponding peptides, H1S and H2S, and confirmed by circular dichroism spectroscopy that they can assume alpha-helical conformation. Oxidative cross-linking of amino-terminal cysteines generated specific covalent oligomers, indicating that separately H1S forms trimers and H2S tetramers. Upon mixing, covalent heterotetramers were formed with a preferred stoichiometry of 2 H1S and 2 H2S peptides. These results suggest that the stalk segments of the receptor subunits oligomerize to constitute an alpha-helical coiled coil stalk on top of which the carbohydrate binding domains are exposed for ligand binding. We propose that the functional asialoglycoprotein receptor is a 2:2 heterotetramer.