Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Basal membrane proteins

The review summarizes recent achievements in biochemistry of basal lamina which is highly specialized structural element of extracellular matrix. Structural and functional characteristics of main basal lamina proteins are reviewed including collagen type IV, laminin, nidogen, and heparan sulfate-containing proteoglycans. Special attention is paid to characteristics of structure and biochemical composition of renal glomerular basal lamina which is one of the main components of renal filtration barrier. Possible methods of investigation and characterization of new basal lamina proteins are discussed which help in understanding of biochemical composition of this structure.     

Detergent-mediated reconstitution of membrane proteins

The efficiency of reconstitution of the lactose transport protein (LacS) of Streptococcus thermophilus is markedly higher with Triton X-100 than with other detergents commonly employed to mediate the membrane insertion. To rationalize these differences, the lipid/detergent structures that are formed in the reconstitution process were studied by cryotransmission electron microscopy. Surprisingly, the two nonionic detergents Triton X-100 and n-dodecyl beta-D-maltoside (DDM) affected the liposome structures in a completely different manner. Preformed liposomes titrated with Triton X-100 maintained their bilayer structure far beyond the onset of solubilization, and transport activity was maximal when LacS was inserted into these structures. With DDM the vesicular structures were already disrupted at the onset of solubilization and these membrane sheets were converted into long threadlike micelles at higher DDM to lipid ratios. Triton X-100 allowed the protein to be reconstituted with the hydrophilic surface exposed to the outside, whereas LacS was incorporated randomly when DDM was used. These differences in orientation are readily explained by the different lipid-detergent structures formed by Triton X-100 and DDM. The orientation of the reconstituted LacS protein is a critical factor for the activity of the protein as the kinetics of translocation is very different for opposite directions of transport.     

Helix packing in membrane proteins

A survey of 45 transmembrane (TM) helices and 88 helix packing interactions in three independent transmembrane protein structures reveals the following features. (1) Helix lengths range from 14 to 36 residues with an average length of 26.4 residues. There is a preference for lengths greater than 20 residues. (2) The helices are tilted with respect to the bilayer normal by an average of 21 degrees, but there is a decided preference for smaller tilt angles. (3) The distribution of helix packing angles is very different than for soluble proteins. The most common packing angles for TM helices are centered around +20 degrees while for soluble proteins packing angles of around -35 degrees are the most prevalent. (4) The average distance of closest approach is 9.6 A, which is the same as soluble proteins. (5) There is no preference for the positioning of the point of closest approach along the length of the helices. (6) It is almost a rule that TM helices pack against neighbors in the sequence. Of the 37 helices that have a sequence neighbor, 36 of them are in significant contact with a neighbor. (7) An antiparallel orientation is more prevalent than a parallel orientation and antiparallel interactions are more intimate on average. The general features of helix bundle membrane protein architecture described in this survey should prove useful in the modeling of helix bundle transmembrane proteins.     

Evidence from normal and degenerating photoreceptors that two outer segment integral membrane proteins have separate transport pathways

Detachment of the neural retina from the retinal pigment epithelium induces photoreceptor degeneration. We studied the effects of this degeneration on the localization of two photoreceptor outer segment-specific integral membrane proteins, opsin and peripherin/rds, in rod photoreceptors. Results from laser scanning confocal microscopic and electron microscopic immunolocalization demonstrate that these two proteins, normally targeted to the newly-forming discs of the outer segments, accumulate in different sub-cellular compartments during photoreceptor degeneration: opsin immunolabeling increases throughout the photoreceptor cell's plasma membrane, while peripherin/rds immunolabeling occurs within cytoplasmic vesicles. The simplest hypothesis to explain our results is that these proteins are transported in different post-Golgi transport vesicles and separately inserted into the plasma membrane. More complex mechanisms involve having the two co-transported and then opsin finds its way into the plasma membrane but peripherin/rds does not, remaining behind in vesicles. Alternatively, both insert into the plasma membrane but peripherin/rds is recycled into cytoplasmic vesicles. We believe the data most strongly supports the first possibility. Although the transport pathways for these proteins have not been fully characterized, the presence of peripherin/rds-positive vesicles adjacent to the striated rootlet suggests a transport role for this cytoskeletal element. The accumulation of these proteins in photoreceptors with degenerated outer segments may also indicate that their rate of synthesis has exceeded the combined rates of their incorporation into newly forming outer segment disc membranes and their degradation. The accumulation may also provide a mechanism for rapid recovery of the outer segment following retinal reattachment and return of the photoreceptor cell to an environment favorable to outer segment regeneration.     

Two-dimensional separation of erythrocyte membrane proteins

1). Erythrocyte membrane proteins eluted with Triton X-100 or dilute EDTA have been separated two-dimensionally by isoelectric focusing in polyacrylamide gels containing 1 percent Triton X-100 plus 8 M urea, followed by electrophoresis using sodium dodecyl sulfate. Characteristic patterns, consistent among 40 healthy donors, were obtained. 2. The resulting patterns contain at least 30 components. The "spectrin" components (sodium dodecyl sulfate Bands 1 and 2) focus in the same pH range. Other membrane components giving single bands in sodium dodecyl sulfate electrophoresis appear to be heterogeneous. 3. Triton X-100, but not EDTA, extracts the principal membrane glycoproteins and the major "intrinsic" protein. Otherwise, proteins preferentially eluted by EDTA extract poorly with Triton X-100 and vice versa. 4. Membrane glycoproteins migrate anodally during electrofocusing and can be purified in a simple, one-step procedure.     

Curvature-mediated interactions between membrane proteins

Membrane proteins can deform the lipid bilayer in which they are embedded. If the bilayer is treated as an elastic medium, then these deformations will generate elastic interactions between the proteins. The interaction between a single pair is repulsive. However, for three or more proteins, we show that there are nonpairwise forces whose magnitude is similar to the pairwise forces. When there are five or more proteins, we show that the nonpairwise forces permit the existence of stable protein aggregates, despite their pairwise repulsions.     

Predicting the topology of eukaryotic membrane proteins

We show that the so-called 'positive inside' rule, i.e. the observation that positively charged amino acids tend to be more prevalent in cytoplasmic than in extra-cytoplasmic segments in transmembrane proteins [von Heijne, G. (1986) EMBO J. 5, 3021-3027], seems to hold for all polar segments in multi-spanning eukaryotic membrane proteins irrespective of their position in the sequence and hence can be used in conjunction with hydrophobicity analysis to predict their transmembrane topology. Further, as suggested by others, we confirm that the net charge difference across the first transmembrane segment correlates well with its orientation [Hartmann, E., Rapoport, T. A. and Lodish H. F. (1989) Proc. Natl Acad. Sci. USA 86, 5786-5790], and that the overall amino-acid composition of long polar segments can also be used to predict their cytoplasmic or extra-cytoplasmic location [Nakashima, H. and Nishikawa, K. (1992) FEBS Lett. 303, 141-146]. We present an approach to the topology prediction problem for eukaryotic membrane proteins based on a combination of these methods.     

Oxidative cross-linking of plasma membrane arabinogalactan proteins

STUDY OBJECTIVE: Endotoxin is a powerful trigger of systemic inflammation. Since cardiac surgery exposes patients to endotoxemia, this study was set up to define the relationship between preoperative endogenous endotoxin immune status, gut perfusion, and outcome following cardiac valve replacement surgery. DESIGN: Observational study. SETTING: University hospital. PATIENTS: Fifty-nine consecutive patients undergoing cardiac valve replacement. MEASUREMENTS AND MAIN RESULTS: Blood was assayed for IgG and IgM endotoxin core antibody (EndoCAb) levels preoperatively, immediately postoperatively, and at 4 h and 24 h postoperatively. Intraoperative gut mucosal perfusion was assessed using gastric tonometry. Complications were assessed for groups above and below the median EndoCAb value of a healthy population (100 median units micro/mL). Of the 59 patients, 12 developed at least one of a set of predefined complications. Of these 12, all had preoperative levels of IgM EndoCAb below 100 MU/mL (p<0.025). Eleven had IgG EndoCAb levels below 100 MU/mL (0.05相似文献   

Preparative two-dimensional gel electrophoresis of membrane proteins

Electrophoretic techniques, and especially two-dimensional gel electrophoresis (2-DE), have provided an indispensable set of tools for the separation of complex protein mixtures as well as for the identification of protein-protein interactions. Nevertheless, after its introduction more than twenty years ago and even with recent technical developments, the separation of integral and peripheral membrane proteins, in amounts sufficient for microsequencing, is still a difficult task. Lipids present in the membrane as well as the low solubility of hydrophobic membrane proteins result in protein aggregation both on the sample application point and on isoelectric focusing. As a consequence many proteins do not enter the first or second dimension of 2-DE. Here we describe the modification of a protocol using a combination of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), chaotropic agents (thiourea, urea), Tris base and reducing agents (1,4-dithioerythritol) to improve solubilization of integral and peripheral membrane proteins. Preparative amounts of membrane proteins (up to 2 mg) were loaded during reswelling of dry immobilized pH gradients and the resulting Coomassie staining patterns were largely superimposable with silver-stained gels obtained from identical samples (4 microg). This indicates that the recovery of proteins from the sample is not significantly compromised by the scale-up procedure. A direct application of this method for the characterization and identification of membrane proteins from cellular organelles is described in another paper in this issue (I. Fialka et al., Electrophoresis 1997, 18, 2582-2590).     

Targeting and retention of Golgi membrane proteins

Recent cloning of genes encoding membrane proteins of the Golgi complex has allowed investigation of protein targeting to this organelle. Targeting signals have been identified in three glycosyltransferases, a viral envelope protein and several proteins of the trans-Golgi network. Interestingly, the targeting signals for membrane proteins of the Golgi stacks seem to be contained in transmembrane domains. Information in the cytoplasmic tails is required for the targeting of trans-Golgi network proteins. Mechanisms involving both retention and retrieval have been invoked.     

Separation of early steps in endocytic membrane transport

We describe a simple subcellular fractionation scheme aimed at separating early endosomes from the plasma membrane in view of studying the possible arrival of plasma membrane-bound toxins, proteins or other extracellular ligands in endosomes. Plasma membrane proteins were labeled with the impermeable reagent sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC) biotin at 4 degrees C. In a separate set of cells, early endosomes were labeled by internalization of horseradish peroxidase from the medium for 5 min. The first step of the purification, which consists of a step sucrose gradient, led to three fractions, respectively: enriched in biosynthetic membranes (interface 3), in plasma membrane and early endosomes (interface 2), and in late endosomes (interface 1). The second step, in which interface 2 was loaded at the bottom of a 17% Percoll gradient, led to the separation of the plasma membrane, including caveolae and cholesterol-glycolipid rafts, from early endosomes. Western blot analysis of the fractions from the Percoll gradient showed that the transferrin receptor, the small GTPases rab5 and Arf6, as well as annexin II were present both at the plasma membrane and in early endosomes, whereas the caveolar marker caveolin, 1co, migrated only with the biotinylated plasma membrane proteins. We used this fractionation procedure to show that the pore-forming toxin aerolysin does not reach the endocytic compartments of baby hamster kidney (BHK) cells. The procedure should be generally useful in rapidly determining whether extracellular proteins or ligands reach endosomes.     

The immunochemical approach to the characterization of membrane proteins. Human erythrocyte membrane proteins analysed as a model system

1. Crossed immunoelectrophoresis was used for extensive characterization of individual proteins of human erythrocyte membranes solubilized in non-ionic detergent. 2. The precipitates were assigned to extrinsic or intrinsic proteins. 3. Four glycoproteins were identified by their lectin binding behaviour, whilst five proteins were affected by neuraminidase, indicating them to be sialoglycoproteins. 4. Enzymatic activity is retained in the solubilized system and the presence of acetylcholinesterase and an ATPase was demonstrated. The formation of phosphorylated membrane proteins on incubation with [32P]ATP was demonstrated by autoradiography on the immunoelectrophoresis plates. 5. Five proteins located on the outer cell surface were identified by antibody binding to intact cells. These same proteins were degraded by proteolytic enzymes in intact cells but only three of them were labelled by lactoperoxidase-catalysed 125I-iodination. 6. Analysis of erythrocyte membrane proteins using quantitive immunoelectrophoresis yields results concordant with those obtained by dodecyl sulfate-polyacrylamide gel electrophoresis.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

Modelling of zinc transport through a supported liquid membrane

The transport of zinc (II) from an aqueous solution containing zinc (II), iron (II), calcium (II) and magnesium (II) through supported liquid membrane using di-2-ethylhexyl phosphoric acid dissolved in kerosene as a mobile carrier was studied. The effects of temperature, rate of feed and stripping phase and concentration of stripping phase on the mass transfer coefficients of aqueous boundary layers and membrane were studied. A transport rate model has been derived taking into account diffusion through the feed side aqueous boundary layer, diffusion of carrier–zinc complex through the supported liquid membrane and diffusion through the stripping side aqueous boundary layer as simultaneous controlling factors. The mass transfer coefficient data of the side of the feed phase were correlated in the form of Sh = 0.0047 Re^1.349 Sc^0.3333. This correlation was used to calculate the mass transfer coefficient of the aqueous film at the side of the stripping phase. For some parameters and their levels, the mass transfer coefficients, k_f, k_m and k_s (m s^− 1), were calculated.