Secretagogue-triggered Transfer of Membrane Proteins from Neuroendocrine Secretory Granules to Synaptic-like Microvesicles

The membrane proteins of all regulated secretory organelles (RSOs) recycle after exocytosis. However, the recycling of those membrane proteins that are targeted to both dense core granules (DCGs) and synaptic-like microvesicles (SLMVs) has not been addressed. Since neuroendocrine cells contain both RSOs, and the recycling routes that lead to either organelle overlap, transfer between the two pools of membrane proteins could occur during recycling. We have previously demonstrated that a chimeric protein containing the cytosolic and transmembrane domains of P-selectin coupled to horseradish peroxidase is targeted to both the DCG and the SLMV in PC12 cells. Using this chimera, we have characterized secretagogue-induced traffic in PC12 cells. After stimulation, this chimeric protein traffics from DCGs to the cell surface, internalizes into transferrin receptor (TFnR)-positive endosomes and thence to a population of secretagogue-responsive SLMVs. We therefore find a secretagogue-dependent rise in levels of HRP within SLMVs. In addition, the levels within SLMVs of the endogenous membrane protein, synaptotagmin, as well as a green fluorescent protein-tagged version of vesicle-associated membrane protein (VAMP)/synaptobrevin, also show a secretagogue-dependent increase.

GERp95, a Membrane-associated Protein that Belongs to a Family of Proteins Involved in Stem Cell Differentiation

A panel of mAbs was elicited against intracellular membrane fractions from rat pancreas. One of the antibodies reacted with a 95-kDa protein that localizes primarily to the Golgi complex or the endoplasmic reticulum (ER), depending on cell type. The corresponding cDNA was cloned and sequenced and found to encode a protein of 97.6 kDa that we call GERp95 (Golgi ER protein 95 kDa). The protein copurifies with intracellular membranes but does not contain hydrophobic regions that could function as signal peptides or transmembrane domains. Biochemical analysis suggests that GERp95 is a cytoplasmically exposed peripheral membrane protein that exists in a protease-resistant complex. GERp95 belongs to a family of highly conserved proteins in metazoans and Schizosaccharomyces pombe. It has recently been determined that plant and Drosophila homologues of GERp95 are important for controlling the differentiation of stem cells (Bohmert et al., 1998; Cox et al., 1998; Moussian et al., 1998). In Caenorhabditis elegans, there are at least 20 members of this protein family. To this end, we have used RNA interference to show that the GERp95 orthologue in C. elegans is important for maturation of germ-line stem cells in the gonad. GERp95 and related proteins are an emerging new family of proteins that have important roles in metazoan development. The present study suggests that these proteins may exert their effects on cell differentiation from the level of intracellular membranes.

Cholesterol-independent Targeting of Golgi Membrane Proteins in Insect Cells

Distinct lipid compositions of intracellular organelles could provide a physical basis for targeting of membrane proteins, particularly where transmembrane domains have been shown to play a role. We tested the possibility that cholesterol is required for targeting of membrane proteins to the Golgi complex. We used insect cells for our studies because they are cholesterol auxotrophs and can be depleted of cholesterol by growth in delipidated serum. We found that two well-characterized mammalian Golgi proteins were targeted to the Golgi region of Aedes albopictus cells, both in the presence and absence of cellular cholesterol. Our results imply that a cholesterol gradient through the secretory pathway is not required for membrane protein targeting to the Golgi complex, at least in insect cells.

Organization of G Proteins and Adenylyl Cyclase at the Plasma Membrane

There is mounting evidence for the organization and compartmentation of signaling molecules at the plasma membrane. We find that hormone-sensitive adenylyl cyclase activity is enriched in a subset of regulatory G protein-containing fractions of the plasma membrane. These subfractions resemble, in low buoyant density, structures of the plasma membrane termed caveolae. Immunofluorescence experiments revealed a punctate pattern of G protein α and β subunits, consistent with concentration of these proteins at distinct sites on the plasma membrane. Partial coincidence of localization of G protein α subunits with caveolin (a marker for caveolae) was observed by double immunofluorescence. Results of immunogold electron microscopy suggest that some G protein is associated with invaginated caveolae, but most of the protein resides in irregular structures of the plasma membrane that could not be identified morphologically. Because regulated adenylyl cyclase activity is present in low-density subfractions of plasma membrane from a cell type (S49 lymphoma) that does not express caveolin, this protein is not required for organization of the adenylyl cyclase system. The data suggest that hormone-sensitive adenylyl cyclase systems are localized in a specialized subdomain of the plasma membrane that may optimize the efficiency and fidelity of signal transduction.

Pex17p Is Required for Import of Both Peroxisome Membrane and Lumenal Proteins and Interacts with Pex19p and the Peroxisome Targeting Signal–Receptor Docking Complex in Pichia pastoris

Pichia pastoris PEX17 was cloned by complementation of a peroxisome-deficient strain obtained from a novel screen for mutants disrupted in the localization of a peroxisomal membrane protein (PMP) reporter. PEX17 encodes a 267-amino-acid protein with low identity (18%) to the previously characterized Saccharomyces cerevisiae Pex17p. Like ScPex17p, PpPex17p contains a putative transmembrane domain near the amino terminus and two carboxyl-terminal coiled-coil regions. PpPex17p behaves as an integral PMP with a cytosolic carboxyl-terminal domain. pex17Δ mutants accumulate peroxisomal matrix proteins and certain integral PMPs in the cytosol, suggesting a critical role for Pex17p in their localization. Peroxisome remnants were observed in the pex17Δ mutant by morphological and biochemical means, suggesting that Pex17p is not absolutely required for remnant formation. Yeast two-hybrid analysis demonstrated that the carboxyl terminus of Pex19p was required for interaction with Pex17p lacking the carboxyl-terminal coiled-coil domains. Biochemical evidence confirmed the interaction between Pex19p and Pex17p. Additionally, Pex17p cross-linked to components of the peroxisome targeting signal–receptor docking complex, which unexpectedly contained Pex3p. Our evidence suggests the existence of distinct subcomplexes that contain separable pools of Pex3p, Pex19p, Pex17p, Pex14p, and the peroxisome targeting signal receptors. These distinct pools may serve different purposes for the import of matrix proteins or PMPs.

A general method for determining helix packing in membrane proteins in situ: Helices I and II are close to helix VII in the lactose permease of Escherichia coli

It was previously shown that coexpression of the lactose permease of Escherichia coli in two contiguous fragments leads to functional complementation. We demonstrate here that site-directed thiol crosslinking of coexpressed permease fragments can be used to determine helix proximity in situ without the necessity of purifying the permease. After coexpression of the six N-terminal (N6) and six C-terminal (C6) transmembrane helices, each with a single Cys residue, crosslinking was carried out in native membranes and assessed by the mobility of anti-C-terminal-reactive polypeptides on immunoblots. A Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 28 or 29 (helix I), but not with a Cys residue at position 27, which is on the opposite face of helix I, thereby indicating that the face of helix I containing Pro-28 and Phe-29 is close to helix VII. Similarly, a Cys residue at position 242 or 245 (helix VII) forms a disulfide with a Cys residue at either position 52 or 53 (helix II), but not with a Cys residue at position 54. Furthermore, low-efficiency crosslinking is observed between a Cys residue at position 52 or 53 and a Cys residue at position 361 (helix XI). The results indicate that helix VII lies in close proximity to both helices I and II and that helix II is also close to helix XI. The method should be applicable to a number of different polytopic membrane proteins.

Lipidic cubic phases: A novel concept for the crystallization of membrane proteins

Understanding the mechanisms of action of membrane proteins requires the elucidation of their structures to high resolution. The critical step in accomplishing this by x-ray crystallography is the routine availability of well-ordered three-dimensional crystals. We have devised a novel, rational approach to meet this goal using quasisolid lipidic cubic phases. This membrane system, consisting of lipid, water, and protein in appropriate proportions, forms a structured, transparent, and complex three-dimensional lipidic array, which is pervaded by an intercommunicating aqueous channel system. Such matrices provide nucleation sites (“seeding”) and support growth by lateral diffusion of protein molecules in the membrane (“feeding”). Bacteriorhodopsin crystals were obtained from bicontinuous cubic phases, but not from micellar systems, implying a critical role of the continuity of the diffusion space (the bilayer) on crystal growth. Hexagonal bacteriorhodopsin crystals diffracted to 3.7 Å resolution, with a space group P63, and unit cell dimensions of a = b = 62 Å, c = 108 Å; α = β = 90° and γ = 120°.

Amphipols: Polymers that keep membrane proteins soluble in aqueous solutions

Amphipols are a new class of surfactants that make it possible to handle membrane proteins in detergent-free aqueous solution as though they were soluble proteins. The strongly hydrophilic backbone of these polymers is grafted with hydrophobic chains, making them amphiphilic. Amphipols are able to stabilize in aqueous solution under their native state four well-characterized integral membrane proteins: (i) bacteriorhodopsin, (ii) a bacterial photosynthetic reaction center, (iii) cytochrome b6f, and (iv) matrix porin.

A novel approach for the identification of protein–protein interaction with integral membrane proteins

Protein–protein interaction plays a major role in all biological processes. The currently available genetic methods such as the two-hybrid system and the protein recruitment system are relatively limited in their ability to identify interactions with integral membrane proteins. Here we describe the development of a reverse Ras recruitment system (reverse RRS), in which the bait used encodes a membrane protein. The bait is expressed in its natural environment, the membrane, whereas the protein partner (the prey) is fused to a cytoplasmic Ras mutant. Protein–protein interaction between the proteins encoded by the prey and the bait results in Ras membrane translocation and activation of a viability pathway in yeast. We devised the expression of the bait and prey proteins under the control of dual distinct inducible promoters, thus enabling a rapid selection of transformants in which growth is attributed solely to specific protein–protein interaction. The reverse RRS approach greatly extends the usefulness of the protein recruitment systems and the use of integral membrane proteins as baits. The system serves as an attractive approach to explore novel protein–protein interactions with high specificity and selectivity, where other methods fail.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Ric1p and the Ypt6p GTPase Function in a Common Pathway Required for Localization of Trans-Golgi Network Membrane Proteins

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1Δ) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1Δ cells also harboring end3Δ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1Δ. Further comparison of ric1Δ and ypt6Δ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v- and t-SNAREs, partially suppressed phenotypes of ric1Δ and ypt6Δ cells. SLY1–20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p- and Ypt6p-dependent retrieval to the cis-Golgi network.

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

The targeting pathway of Escherichia coli presecretory and integral membrane proteins is specified by the hydrophobicity of the targeting signal

Previous studies have demonstrated that presecretory proteins such as maltose binding protein (MBP) and outer membrane protein A (OmpA) are targeted to the Escherichia coli inner membrane by the molecular chaperone SecB, but that integral membrane proteins are targeted by the signal recognition particle (SRP). In vitro studies have suggested that trigger factor binds to a sequence near the N terminus of the mature region of OmpA and shunts the protein into the SecB pathway by blocking an interaction between SRP and the signal peptide. By contrast, we have found that the targeting pathway of a protein under physiological conditions is dictated by the composition of its targeting signal. Replacement of the MBP or OmpA signal peptide with the first transmembrane segment of AcrB abolished the dependence on SecB for transport and rerouted both proteins into the SRP targeting pathway. More modest alterations of the MBP signal peptide that simply increase its hydrophobicity also promoted SRP binding. Furthermore, we obtained evidence that SRP has a low affinity for typical signal peptides in vivo. These results imply that different classes of E. coli proteins are targeted by distinct pathways because bacterial SRP binds to a more restricted range of targeting signals than its eukaryotic counterpart.

Epstein–Barr virus latent-infection membrane proteins are palmitoylated and raft-associated: Protein 1 binds to the cytoskeleton through TNF receptor cytoplasmic factors

Epstein–Barr virus encodes integral membrane proteins LMP1 and LMP2A in transformed lymphoblastoid cell lines. We now find that LMP1 associates with the cell cytoskeleton through a tumor necrosis factor receptor-associated factor-interacting domain, most likely mediated by tumor necrosis factor receptor-associated factor 3. LMP1 is palmitoylated, and the transmembrane domains associate with lipid rafts. Mutation of LMP1 cysteine-78 abrogates palmitoylation but does not affect raft association or NF-κB or c-Jun N-terminal kinase activation. LMP2A also associates with rafts and is palmitoylated but does not associate with the cell cytoskeleton. The associations of LMP1 and LMP2A with rafts and of LMP1 with the cell cytoskeleton are likely to effect interactions with cell proteins involved in shape, motility, signal transduction, growth, and survival.