Topology of polytopic membrane protein subdomains is dictated by membrane phospholipid composition

In Escherichia coli, the major cytoplasmic domain (C6) of the polytopic membrane protein lactose permease (LacY) is exposed to the opposite side of the membrane from a neighboring periplasmic domain (P7). However, these domains are both exposed on the periplasmic side of the membrane in a mutant of E.coli lacking phosphatidylethanolamine (PE) wherein LacY only mediates facilitated transport. When purified LacY was reconstituted into liposomes lacking PE or phosphatidylcholine (PC), C6 and P7 were on the same side of the bilayer. In liposomes containing PE or PC, C6 and P7 were on opposite sides of the bilayer. Only the presence of PE in the liposomes restored active transport function of LacY as opposed to restoration of only facilitated transport function in the absence of PE. These results were the same for LacY purified from PE-containing or PE-lacking cells, and are consistent with the topology and function of LacY assembled in vivo. Therefore, irrespective of the mechanism of membrane insertion, the subdomain topological orientation and function of LacY are determined primarily by membrane phospholipid composition.     

Left‐right asymmetry in the chick embryo requires core planar cell polarity protein Vangl2

Consistent left‐right patterning is a fascinating and biomedically important problem. In the chick embryo, it is not known how cells determine their position (left or right) relative to the primitive streak, which is required for subsequent asymmetric gene expression cascades. We show that the subcellular localization of Vangl2, a core planar cell polarity (PCP) protein, is consistently polarized, giving cells in the blastoderm a vector pointing toward the primitive streak. Moreover, morpholino‐mediated loss‐of‐function of Vangl2 by electroporation into chicks at very early stages randomizes the normally left‐sided expression of Sonic hedgehog. Strikingly, Vangl2 morpholinos also induce a desynchronization of asymmetric gene expression within the left and right domains of Hensen's node. These data reveal the existence of polarized planar cell polarity protein localization in gastrulating chick and demonstrate that the PCP pathway is functionally required for normal asymmetry in the chick upstream of Sonic hedgehog. These data suggest a new and widely applicable class of models for the spread and coordination of left‐right patterning information in the embryonic blastoderm. genesis 47:719–728, 2009. © 2009 Wiley‐Liss, Inc.     

Differential activities of the core planar polarity proteins during Drosophila wing patterning

During planar polarity patterning of the Drosophila wing, a "core" group of planar polarity genes has been identified which acts downstream of global polarity cues to locally coordinate cell polarity and specify trichome production at distal cell edges. These genes encode protein products that assemble into asymmetric apicolateral complexes that straddle the proximodistal junctional region between adjacent cells. We have carried out detailed genetic analysis experiments, analysing the requirements of each complex component for planar polarity patterning. We find that the three transmembrane proteins at the core of the complex, Frizzled, Strabismus and Flamingo, are required earliest in development and are the only components needed for intercellular polarity signalling. Notably, cells that lack both Frizzled and Strabismus are unable to signal, revealing an absolute requirement for both proteins in cell-cell communication. In contrast the cytoplasmic components Dishevelled, Prickle and Diego are not needed for intercellular communication. These factors contribute to the cell-cell propagation of polarity, most likely by promotion of intracellular asymmetry. Interestingly, both local polarity propagation and trichome placement occur normally in mutant backgrounds where asymmetry of polarity protein distribution is undetectable, suggesting such asymmetry is not an absolute requirement for any of the functions of the core complex.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

Transmembrane topology of mammalian planar cell polarity protein Vangl1

Vangl1 and Vangl2 are membrane proteins that play an important role in neurogenesis, and Vangl1/Vangl2 mutations cause neural tube defects in mice and humans. At the cellular level, Vangl proteins regulate the establishment of planar cell polarity (PCP), a process requiring membrane assembly of asymmetrically distributed multiprotein complexes that transmit polarity information to neighboring cells. The membrane topology of Vangl proteins and the protein segments required for structural and functional aspects of multiprotein membrane PCP complexes is unknown. We have used epitope tagging and immunofluorescence to establish the secondary structure of Vangl proteins, including the number, position, and polarity of transmembrane domains. Antigenic hemagglutinin A (HA) peptides (YYDVPDYS) were inserted in predicted intra- or extracellular loops of Vangl1 at positions 18, 64, 139, 178, 213, and 314, and individual mutant variants were stably expressed at the membrane of MDCK polarized cells. The membrane topology of the exofacial HA tag was determined by a combination of immunofluorescence in intact (extracellular epitopes) and permeabilized (intracellular epitopes) cells and by surface labeling. Results indicate that Vangl proteins have a four-transmembrane domain structure with the N-terminal portion (HA18 and HA64) and the large C-terminal portion (HA314) of the protein being intracellular. Topology mapping and hydropathy profiling show that the loop delineated by TMD1-2 (HA139) and TMD3-4 (HA213) is extracellular while the segment separating predicted TMD2-3 (HA178) is intracellular. This secondary structure reveals a compact membrane-associated portion with very short predicted extra- and intracellular loops, while the protein harbors a large intracellular domain.     

Clustering induces redistribution of syndecan-4 core protein into raft membrane domains

Syndecan-4 is a heparan sulfate-carrying core protein that has been directly implicated in fibroblast growth factor 2 (FGF2) signaling. Recent studies have suggested that many signaling proteins localize to the raft compartment of the plasma cell membrane. To establish whether syndecan-4 is present in the raft compartment, we have studied the distribution of the core protein and an Fc receptor (FcR)-syndecan-4 chimera prior to and following clustering with FGF2 or antibodies. Whereas unclustered syndecan-4 was present predominantly in the non-raft membrane compartment, clustering induced extensive syndecan-4 redistribution to the rafts as demonstrated by the sucrose gradient centrifugation and life confocal microscopy. Although syndecan-4 and caveolin-1 moved in tandem, syndecan-4 was not present in caveolae, a major subset of raft compartments. We conclude that syndecan-4 clustering induces its redistribution to the non-caveolae raft compartment. This process may play an important role in syndecan-4-mediation of FGF2 signaling.     

The machinery of membrane protein assembly

The SecY (bacteria) and Sec61 (eukaryotes) translocon complexes, or protein-conducting channels, work in concert with bound ribosomes to insert proteins into membranes during the first step of membrane protein assembly. The crystallographic structure of an archaeal SecY translocon provides dramatic new insights into the mechanism of translocon function. This structure suggests an explanation for how the translocon can aid in establishing membrane protein topology via the positive-inside rule. The folding of membrane proteins may begin in the ribosome exit tunnel, before entering the translocon, according to cryo-electron microscopy and biophysical studies.     

The turnover of protein in discrete areas of rat brain

1. Rats were injected serially with [(14)C]glucose to obtain a constant specific radioactivity of brain amino acids. Measurements with this system for periods of up to 8h gave an apparent mean half-life for protein in whole brain of 85h (indicating the presence of a protein fraction with much more rapid turnover than this). 2. The half-lives of proteins in the granule-cell, molecular and white-matter layers of cerebellum were also determined. These had values of 33, 59 and 136h respectively. In addition, the incorporation into protein in six layers of the cerebral cortex, subjacent white matter and five layers of Ammon's horn was studied. All cell-body layers incorporated amino acids at about the same rate irrespective of location, and these rates were considerably higher than those for incorporation into proteins in areas rich in dendrites or fibre tracts. 3. A new method for measuring small amounts of glutamate with a cyclic enzyme system is presented.     

The assembly of membrane proteins into complexes

Protein complexes are a fundamental aspect of life in a membrane. It is therefore important to understand which proteins are assembled, and how the process of assembly is coordinated. To this end, a number of themes have emerged from the literature in recent years: first, membrane proteins assemble in an ordered, rather than a stochastic manner; second, they require chaperones to prevent unwanted interactions/aggregation; and third, they can be assembled into existing complexes. As these recurrent themes have emerged from studies on disparate complexes, they provide a general framework to understand the assembly of membrane proteins.     

Polarized membrane traffic and cell polarity development is dependent on dihydroceramide synthase-regulated sphinganine turnover

Sphingoid bases have been implicated in various cellular processes including cell growth, apoptosis and cell differentiation. Here, we show that the regulated turnover of sphingoid bases is crucial for cell polarity development, i.e., the biogenesis of apical plasma membrane domains, in well-differentiated hepatic cells. Thus, inhibition of dihydroceramide synthase or sphinganine kinase activity with fumonisin B1 or N,N-dimethylsphingosine, respectively, dramatically perturbs cell polarity development, which is due to increased levels of sphinganine. Consistently, reduction of free sphinganine levels stimulates cell polarity development. Moreover, dihydroceramide synthase, the predominant enzyme responsible for sphinganine turnover, is a target for cell polarity stimulating cAMP/protein kinase A (PKA) signaling cascades. Indeed, electrospray ionization tandem mass spectrometry analyses revealed a significant reduction in sphinganine levels in cAMP/PKA-stimulated cells. These data suggest that sphinganine turnover is critical for and is actively regulated during HepG2 cell polarity development. Previously, we have identified an apical plasma membrane-directed trafficking pathway from the subapical compartment. This transport pathway, which is part of the basolateral-to-apical transcytotic itinerary, plays a crucial role in apical plasma membrane biogenesis. Here, we show that, as a part of the underlying mechanism, the inhibition of dihydroceramide synthase activity and ensuing increased sphinganine levels specifically perturb the activation of this particular pathway in the de novo apical membrane biogenesis.     

Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V‐ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co‐localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E‐Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V‐ATPase subunits. By contrast, the V‐ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.     

Statistical mechanics of integral membrane protein assembly

During the synthesis of integral membrane proteins (IMPs), the hydrophobic amino acids of the polypeptide sequence are partitioned mostly into the membrane interior and hydrophilic amino acids mostly into the aqueous exterior. Using a many-body statistical mechanics model, we analyze the minimum free energy state of polypeptide sequences partitioned into α-helical transmembrane (TM) segments and the role of thermal fluctuations. Results suggest that IMP TM segment partitioning shares important features with general theories of protein folding. For random polypeptide sequences, the minimum free energy state at room temperature is characterized by fluctuations in the number of TM segments with very long relaxation times. Moreover, simple assembly scenarios do not produce a unique number of TM segments due to jamming phenomena. On the other hand, for polypeptide sequences corresponding to actual IMPs, the minimum free energy structure with the wild-type number of segments is free of number fluctuations due to an anomalously large gap in the energy spectrum. Now, simple assembly scenarios do reproduce the minimum free energy state without jamming. Finally, we find a threshold number of random point mutations where the size of the anomalous gap is reduced to the point that the wild-type ground state is destabilized and number fluctuations reappear.     

Dynamics of ESCRT protein recruitment during retroviral assembly

The ESCRT (endosomal sorting complex required for transport) complexes and associated proteins mediate membrane scission reactions, such as multivesicular body formation, the terminal stages of cytokinesis and retroviral particle release. These proteins are believed to be sequentially recruited to the site of membrane scission, and then complexes are disassembled by the ATPase Vps4A. However, these events have never been observed in living cells, and their dynamics are unknown. By quantifying the recruitment of several ESCRT and associated proteins during the assembly of two retroviruses, we show that Alix progressively accumulated at viral assembly sites, coincident with the accumulation of the main viral structural protein, Gag, and was not recycled after assembly. In contrast, ESCRT-III and Vps4A were transiently recruited only when the accumulation of Gag was complete. These data indicate that the rapid and transient recruitment of proteins that act late in the ESCRT pathway and carry out membrane fission is triggered by prior and progressive accumulation of proteins that bridge viral proteins and the late-acting ESCRT proteins.     

Structural and biochemical insights into MLL1 core complex assembly

Histone H3 Lys-4 methylation is predominantly catalyzed by a family of methyltransferases whose enzymatic activity depends on their interaction with a three-subunit complex composed of WDR5, RbBP5, and Ash2L. Here, we report that a segment of 50 residues of RbBP5 bridges the Ash2L C-terminal domain to WDR5. The crystal structure of WDR5 in ternary complex with RbBP5 and MLL1 reveals that both proteins binds peptide-binding clefts located on opposite sides of WDR5's β-propeller domain. RbBP5 engages in several hydrogen bonds and van der Waals contacts within a V-shaped cleft formed by the junction of two blades on WDR5. Mutational analyses of both the WDR5 V-shaped cleft and RbBP5 residues reveal that the interactions between RbBP5 and WDR5 are important for the stimulation of MLL1 methyltransferase activity. Overall, this study provides the structural basis underlying the formation of the WDR5-RbBP5 subcomplex and further highlight the crucial role of WDR5 in scaffolding the MLL1 core complex.     

Lipids and topological rules governing membrane protein assembly

Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Peroxisome assembly: matrix and membrane protein biogenesis

Mitochondria are dynamic organelles whose functional integrity requires a coordinated supply of proteins and phospholipids. Defined functions of specific phospholipids, like the mitochondrial signature lipid cardiolipin, are emerging in diverse processes, ranging from protein biogenesis and energy production to membrane fusion and apoptosis. The accumulation of phospholipids within mitochondria depends on interorganellar lipid transport between the endoplasmic reticulum (ER) and mitochondria as well as intramitochondrial lipid trafficking. The discovery of proteins that regulate mitochondrial membrane lipid composition and of a multiprotein complex tethering ER to mitochondrial membranes has unveiled novel mechanisms of mitochondrial membrane biogenesis.     

Lateral plasma membrane compartmentalization links protein function and turnover

Biological membranes organize their proteins and lipids into nano‐ and microscale patterns. In the yeast plasma membrane (PM), constituents segregate into a large number of distinct domains. However, whether and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, physiological function, and endocytic turnover. Using the methionine permease Mup1 as model system, we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner protein Nce102, and signaling through TORC2. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM. Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.