Unusual Membrane Topology Research Articles

Vps68 cooperates with ESCRT-III in intraluminal vesicle formation.

The endosomal sorting complex required for transport (ESCRT)-III mediates budding and abscission of intraluminal vesicles (ILVs) into multivesicular endosomes. To further define the role of the yeast ESCRT-III-associated protein Mos10 (also known as Vps60) in ILV formation, we screened for new interaction partners by using stable isotope labeling of amino acids in cell culture (SILAC) and mass spectrometry. Here, we focused on the newly identified interaction partner Vps68. Our data suggest that Vps68 cooperates with ESCRT-III in ILV formation. The deletion of VPS68 caused a sorting defect similar to that of the SNF7 deletion strain when the cargo load was high. The composition of ESCRT-III was altered, the level of core components was higher and the level of associated proteins was lower in the VPS68 deletion strain. Our data further indicate that at some point in the functional cycle of ESCRT-III, Snf7 could be replaced by Mos10. Vps68 has an unusual membrane topology. Two of its potential membrane helices are amphipathic helices that localize to the luminal side of the endosomal membrane. Based on this membrane topology, we propose that Vps68 and ESCRT-III cooperate in the abscission step by weakening the luminal and cytosolic leaflets of the bilayer at the abscission site.

Charged Residues in the Membrane Anchor of the Pestiviral Erns Protein Are Important for Processing and Secretion of Erns and Recovery of Infectious Viruses.

The pestivirus envelope protein Erns is anchored in membranes via a long amphipathic helix. Despite the unusual membrane topology of the Erns membrane anchor, it is cleaved from the following glycoprotein E1 by cellular signal peptidase. This was proposed to be enabled by a salt bridge-stabilized hairpin structure (so-called charge zipper) formed by conserved charged residues in the membrane anchor. We show here that the exchange of one or several of these charged residues reduces processing at the Erns carboxy-terminus to a variable extend, but reciprocal mutations restoring the possibility to form salt bridges did not necessarily restore processing efficiency. When introduced into an Erns-only expression construct, these mutations enhanced the naturally occurring Erns secretion significantly, but again to varying extents that did not correlate with the number of possible salt bridges. Equivalent effects on both processing and secretion were also observed when the proteins were expressed in avian cells, which points at phylogenetic conservation of the underlying principles. In the viral genome, some of the mutations prevented recovery of infectious viruses or immediately (pseudo)reverted, while others were stable and neutral with regard to virus growth.

Membrane orientation and oligomerization of the melanocortin receptor accessory protein 2

The melanocortin receptor accessory protein 2 (MRAP2) plays a pivotal role in the regulation of several G protein-coupled receptors that are essential for energy balance and food intake. MRAP2 loss-of-function results in obesity in mammals. MRAP2 and its homolog MRAP1 have an unusual membrane topology and are the only known eukaryotic proteins that thread into the membrane in both orientations. In this study, we demonstrate that the conserved polybasic motif that dictates the membrane topology and dimerization of MRAP1 does not control the membrane orientation and dimerization of MRAP2. We also show that MRAP2 dimerizes through its transmembrane domain and can form higher-order oligomers that arrange MRAP2 monomers in a parallel orientation. Investigating the molecular details of MRAP2 structure is essential for understanding the mechanism by which it regulates G protein-coupled receptors and will aid in elucidating the pathways involved in metabolic dysfunction.

How the ER Stays in Shape

Understanding the molecular mechanisms that control the architecture of organelles is an area of intense study. In this issue of Cell, Voeltz et al. (2006) report that two membrane proteins, Rtn4a/NogoA and DP1/Yop1p, are responsible for the generation of tubular morphology in the endoplasmic reticulum (ER). The unusual membrane topology of these proteins may directly contribute to ER curvature.

Function of FXYD Proteins, Regulators of Na, K-ATPase

In this short review, we summarize our work on the role of members of the FXYD protein family as tissue-specific modulators of Na, K-ATPase. FXYD1 or phospholemman, mainly expressed in heart and skeletal muscle increases the apparent affinity for intracellular Na(+) of Na, K-ATPase and may thus be important for appropriate muscle contractility. FXYD2 or gamma subunit and FXYD4 or CHIF modulate the apparent affinity for Na(+) of Na, K-ATPase in an opposite way, adapted to the physiological needs of Na(+) reabsorption in different segments of the renal tubule. FXYD3 expressed in stomach, colon, and numerous tumors also modulates the transport properties of Na, K-ATPase but it has a lower specificity of association than other FXYD proteins and an unusual membrane topology. Finally, FXYD7 is exclusively expressed in the brain and decreases the apparent affinity for extracellular K(+), which may be essential for proper neuronal excitability.

Prominin: a story of cholesterol, plasma membrane protrusions and human pathology.

Prominin is the first identified member of a novel family of polytopic membrane proteins conserved throughout the animal kingdom. It has an unusual membrane topology, containing five transmembrane domains and two large glycosylated extracellular loops. In mammals, prominin is expressed in various embryonic and adult epithelial cells, as well as in nonepithelial cells, such as hematopoietic stem cells. At the subcellular level, prominin is selectively localized in microvilli and other plasma membrane protrusions, irrespective of cell type. At the molecular level, prominin specifically interacts with membrane cholesterol and is a marker of a novel type of cholesterol-based lipid 'raft'. A frameshift mutation in the human prominin gene, which results in a truncated protein that is no longer transported to the cell surface, is associated with retinal degeneration. Given that prominin is concentrated in the plasma membrane evaginations at the base of the outer segment of rod photoreceptor cells, which are essential precursor structures in the biogenesis of photoreceptive disks, it is proposed that prominin has a role in the generation of plasma membrane protrusions, their lipid composition and organization and their membrane-to-membrane interactions.

A Yeast Golgi E-type ATPase with an Unusual Membrane Topology

E-type ATPases are involved in many biological processes such as modulation of neural cell activity, prevention of intravascular thrombosis, and protein glycosylation. In this study, we show that a gene of Saccharomyces cerevisiae, identified by similarity to that of animal ectoapyrase CD39, codes for a new member of the E-type ATPase family (Apy1p). Overexpression of Apy1p in yeast cells causes an increase in intracellular membrane-bound nucleoside di- and triphosphate hydrolase activity. The activity is highest with ADP as substrate and is stimulated similarly by Ca (2+), Mg(2+), and Mn(2+). The results also indicate that Apy1p is an integral membrane protein located predominantly in the Golgi compartment. Sequence analysis reveals that Apy1p contains one large NH(2)-terminal hydrophilic apyrase domain, one COOH-terminal hydrophilic domain, and two hydrophobic stretches in the central region of the polypeptide. Although no signal sequence is found at the NH(2)-terminal portion of the protein and no NH(2)-terminal cleavage of the protein is observed, demonstrated by the detection of NH(2)-terminal tagged Apy1p, the NH(2)-terminal domain of Apylp is on the luminal side of the Golgi apparatus, and the COOH-terminal hydrophilic domain binds to the cytoplasmic face of the Golgi membrane. The second hydrophobic stretch of Apy1p is the transmembrane domain. These results indicate that Apylp is a type III transmembrane protein; however, the size of the Apy1p extracytoplasmic NH(2) terminus is much larger than those of other type III transmembrane proteins, suggesting that a novel translocation mechanism is utilized.

The CorA magnesium transporter gene family.

The CorA transport system is the primary Mg2+ influx system of Salmonella typhimurium and Escherichia coli. The CorA protein has no homology to any other known family of proteins. It has an unusual membrane topology, with a large, soluble, highly charged periplasmic N-terminal domain with three transmembrane segments in a shorter, hydrophobic C-terminal domain. Previous phenotypic and molecular data had suggested that this transport system was widespread in the Bacteria. In this report we show that CorA is virtually ubiquitous in the Bacteria and Archaea, forming a distinct family of transport proteins. Genomic sequences to date have revealed at least 22 members of the CorA family in the Bacteria and the Archaea, with 6 more distant members in the yeasts. Only three of the smallest bacterial genomes lack a CorA homologue. Strikingly, phylogenetic analysis does not show clustering by related species or even within kingdom. Several species of Bacteria contain two or even three CorA paralogues. Within species, these paralogues are not closely related, however, and we suggest that they might have distinct transport functions. A multiple alignment suggests three extended consensus regions within the N-terminal soluble domain of CorA, which is predicted to be virtually all alpha-helical. A fourth consensus region includes the last 20 residues of the soluble domain and continues through the entire membrane domain. The first half of this last consensus domain may form an amphipathic alpha-helix that extends from the soluble domain into the first transmembrane segment. The degree of charge in the first transmembrane segment is quite variable, and we suggest that this transport family may include members with only two rather than three transmembrane segments. If so, this would place the N-terminal soluble domain on different sides of the membrane in different members of the family. We suggest that the CorA Mg2+ transport system forms the major Mg2+ uptake system in the Bacteria and Archaea but that some family members may have a function other than Mg2+ transport.

Expression and Characterization of Recombinant Caveolin: PURIFICATION BY POLYHISTIDINE TAGGING AND CHOLESTEROL-DEPENDENT INCORPORATION INTO DEFINED LIPID MEMBRANES

Caveolin, a 22-24-kDa integral membrane protein, is a principal component of caveolar membranes in vivo. Caveolin has been proposed to function as a scaffolding protein to organize and concentrate signaling molecules within caveolae. Because of its unusual membrane topology, both the N- and C-terminal domains of caveolin remain entirely cytoplasmic and are not subject to luminal modifications that are accessible to other integral membrane proteins. Under certain conditions, caveolin also exists in a soluble form as a cytosolic protein in vivo. These properties make caveolin an attractive candidate for recombinant expression in Escherichia coli. Here, we successfully expressed recombinant full-length caveolin in E.coli. A polyhistidine tag was placed at its extreme C terminus for purification by Ni(2+)-nitrilotriacetic acid affinity chromatography. Specific antibody probes demonstrated that recombinant caveolin contained a complete N and C terminus. Recombinant caveolin remained soluble in solutions containing the detergent octyl glucoside and formed high molecular mass oligomers like endogenous caveolin. By electron microscopy, recombinant caveolin homo-oligomers appeared as individual spherical particles that were indistinguishable from endogenous caveolin homo-oligomers visualized by the same technique. As recombinant caveolin behaved as expected for endogenous caveolin, this provides an indication that recombinant caveolin can be used to dissect the structural and functional interaction of caveolin with other protein and lipid molecules in vitro. Recombinant caveolin was efficiently incorporated into lipid membranes as assessed by floatation in sucrose density gradients. This allowed us to use defined lipid components to assess the possible requirements for insertion of caveolin into membranes. Using a purified synthetic form of phosphatidylcholine (1,2-dioleoylphosphorylcholine), we observed that incorporation of caveolin into membranes was cholesterol-dependent; the addition of cholesterol dramatically increased the incorporation of caveolin into these phosphatidylcholine-based membranes by approximately 25-30-fold. This fits well with in vivo studies demonstrating that cholesterol plays an essential role in maintaining the structure and function of caveolae. Further functional analysis of these reconstituted caveolin-containing membranes showed that they were capable of recruiting a soluble recombinant form of G(i)2 alpha. This is in accordance with previous studies demonstrating that caveolin specifically interacts directly with multiple G protein alpha-subunits. Thus, recombinant caveolin incorporated into defined lipid membranes provides an experimental system in which the structure, function, and biogenesis of caveolin-rich membrane domains can be dissected in vitro.

Location and unusual membrane topology of the immunity protein of the Escherichia coli phage T4

The immunity protein (Imm) encoded by the Escherichia coli phage T4 effects exclusion of phage superinfecting cells already infected with T4. The 83-residue polypeptide possesses two long lipophilic areas (from residues 3 to 32 and 37 to 65) interrupted by a hydrophilic stretch including two positively charged residues. The charge distribution of the protein very strongly suggested that it is a plasma membrane protein with the C terminus facing the periplasm. While it could be shown that the expected location was correct, fusions of Imm to alkaline phosphatase or beta-galactosidase showed that the C terminus was at the cytosolic side of the membrane. Also, concerning function, there was almost no structural specificity to this part of the protein. Even removal of the two positively charged residues did not completely abolish function. Evidence suggesting that Imm is associated with the membrane at specific sites is presented. It is proposed that Imm is localized to the membrane with the help of a receptor and that, therefore, it does not follow the established rules for the topology of other membrane proteins. The results also suggest that Imm acts indirectly, possibly by altering the conformation of a component of a phage DNA injection site.

Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation.

The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the M2 protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the M2 topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues to M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation.