A Membrane Topology Model for Human Interferon Inducible Transmembrane Protein 1

InterFeron Inducible TransMembrane proteins 1–3 (IFITM1, IFITM2 and IFITM3) are a family of proteins capable of inhibiting the cellular entry of numerous human and animal viruses. IFITM1-3 are unique amongst the currently described viral restriction factors in their apparent ability to block viral entry. This restrictive property is dependant on the localisation of the proteins to plasma and endosomal membranes, which constitute the main portals of viral entry into cells. The topology of the IFITM proteins within cell membranes is an unresolved aspect of their biology. Here we present data from immunofluorescence microscopy, protease cleavage, biotin-labelling and immuno-electron microscopy assays, showing that human IFITM1 has a membrane topology in which the N-terminal domain resides in the cytoplasm, and the C-terminal domain is extracellular. Furthermore, we provide evidence that this topology is conserved for all of the human interferon-induced IFITM proteins. This model is consistent with that recently proposed for murine IFITM3, but differs from that proposed for murine IFITM1.     

Interferon-induced Transmembrane Protein 3 Is a Type II Transmembrane Protein

The interferon-induced transmembrane (IFITM) proteins are a family of small membrane proteins that inhibit the cellular entry of several genera of viruses. These proteins had been predicted to adopt a two-pass, type III transmembrane topology with an intracellular loop, two transmembrane helices (TM1 and TM2), and extracellular N and C termini. Recent work, however, supports an intramembrane topology for the helices with cytosolic orientation of both termini. Here we determined the topology of murine Ifitm3. We found that the N terminus of Ifitm3 could be stained by antibodies at the cell surface but that this conformation was cell type-dependent and represented a minority of the total plasma membrane pool. In contrast, the C terminus was readily accessible to antibodies at the cell surface and extracellular C termini comprised most or all of those present at the plasma membrane. The addition of a C-terminal KDEL endoplasmic reticulum retention motif to Ifitm3 resulted in sequestration of Ifitm3 in the ER, demonstrating an ER-luminal orientation of the C terminus. C-terminal, but not N-terminal, epitope tags were also degraded within lysosomes, consistent with their luminal orientation. Furthermore, epitope-tagged Ifitm3 TM2 functioned as a signal anchor sequence when expressed in isolation. Collectively, our results demonstrate a type II transmembrane topology for Ifitm3 and will provide insight into its interaction with potential targets and cofactors.     

Mitofilin Is a Transmembrane Protein of the Inner Mitochondrial Membrane Expressed as Two Isoforms

Mitofilin, also known as heart muscle protein, is a recently identified mitochondrial protein. We have isolated two human cDNAs that encode different isoforms of mitofilin. Using reverse PCR, we provide evidence that both isoforms are derived by alternative splicing and encode two proteins of 88 and 90 kDa that are detected in immunoblot analyses with mitofilin-specific antibodies. Immunofluorescence microscopy, fractionating of human osteosarcoma cells, and protease protection experiments with isolated mitochondria and mitoplasts indicate that mitofilin is an integral membrane protein of the inner mitochondrial membrane.³⁵S-labeled mitofilin is transported into isolated yeast mitochondria in a reaction that depends on the membrane potential across the inner mitochondrial membrane (ΔΨ). During mitochondrialin vitroimport, mitofilin is proteolytically processed to the mature protein that is also detected in cellular fractions, indicating that the amino-terminal leader sequence is removed. Sequence analysis and our results suggest that mitofilin is anchored in the inner mitochondrial membrane with an amino-terminal transmembrane domain, while the majority of the protein is extruding into the intermembrane space.     

The Association of ClipR-59 Protein with AS160 Modulates AS160 Protein Phosphorylation and Adipocyte Glut4 Protein Membrane Translocation

ClipR-59 is a membrane-associated protein and has been implicated in membrane signaling and vesicle trafficking. Recently, we have identified ClipR-59 as an Akt-interacting protein, and we have found that, by interacting with Akt, ClipR-59 modulates Akt subcellular compartmentalization and Akt substrate AS160 phosphorylation, thereby promoting Glut4 membrane translocation. Here, we have further investigated the regulatory effects of ClipR-59 on AS160 phosphorylation and subsequent adipocyte glucose transport. Our data showed that ClipR-59 interacted with AS160, which was mediated by the ankyrin repeats of ClipR-59 and regulated by insulin signaling. Moreover, the data also demonstrated that the interaction of ClipR-59 with AS160 was required for ClipR-59 to modulate Glut4 membrane translocation as ΔANK-ClipR-59, an AS160 interaction-defective mutant, failed to promote AS160 phosphorylation, Glut4 membrane translocation, and glucose transport induced by insulin in 3T3-L1 adipocytes. Because ClipR-59 also interacts with Akt and enhances the interaction between Akt and AS160, we suggest that ClipR-59 functions as a scaffold protein to facilitate Akt-mediated AS160 phosphorylation, thereby regulating glucose transport.     

The Transmembrane Protein of the Human Endogenous Retrovirus - K (HERV-K) Modulates Cytokine Release and Gene Expression

Numerous copies of endogenous retroviruses are present in the genome of mammals including man. Although most of them are defective, some, e.g., the human endogenous retroviruses HERV-K, were found to be expressed under certain physiological conditions. For instance, HERV-K is expressed in germ cell tumours and melanomas as well as in the placenta. Most exogenous retroviruses including the human immunodeficiency virus HIV-1 induce severe immunodeficiencies and there is increasing evidence that the transmembrane envelope (TM) proteins of these retroviruses may be involved. We show here that HERV-K particles released from a human teratocarcinoma cell line, a recombinant TM protein and a peptide corresponding to a highly conserved so-called immunosuppressive domain in the TM protein of HERV-K inhibit the proliferation of human immune cells, induce modulation of the expression of numerous cytokines, and modulate the expression of cellular genes as detected by a microarray analysis. The changes in cytokine release and gene expression induced by the TM protein of HERV-K are similar to those found previously induced by the TM protein of HIV-1. These data suggest that the mechanism of immunosuppression may be similar for different retroviruses and that the expression of the TM protein in tumours and in the placenta may suppress immune responses and thus prevent rejection of the tumour and the embryo.     

Calmodulin Modulates Mitogen-Activated Protein Kinase Activation in Response to Membrane Depolarization in PC12 Cells

Abstract: In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca²⁺ influx through voltage-gated Ca²⁺ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca²⁺ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K⁺ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca²⁺ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K⁺ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca²⁺ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Membrane fusion is induced by SNARE complexes that are anchored in both fusion partners. SNAREs zipper up from the N to C terminus bringing the two membranes into close apposition. Their transmembrane domains (TMDs) might be mere anchoring devices, deforming bilayers by mechanical force. Structural studies suggested that TMDs might also perturb lipid structure by undergoing conformational transitions or by zipping up into the bilayer. Here, we tested this latter hypothesis, which predicts that the activity of SNAREs should depend on the primary sequence of their TMDs. We replaced the TMDs of all vacuolar SNAREs (Nyv1, Vam3, and Vti1) by a lipid anchor, by a TMD from a protein unrelated to the membrane fusion machinery, or by artificial leucine-valine sequences. Individual exchange of the native SNARE TMDs against an unrelated transmembrane anchor or an artificial leucine-valine sequence yielded normal fusion activities. Fusion activity was also preserved upon pairwise exchange of the TMDs against unrelated peptides, which eliminates the possibility for specific TMD-TMD interactions. Thus, a specific primary sequence or zippering beyond the SNARE domains is not a prerequisite for fusion. Lipid-anchored Vti1 was fully active, and lipid-anchored Nyv1 permitted the reaction to proceed up to hemifusion, and lipid-anchored Vam3 interfered already before hemifusion. The unequal contribution of proteinaceous TMDs on Vam3 and Nyv1 suggests that Q- and R-SNAREs might make different contributions to the hemifusion intermediate and the opening of the fusion pore. Furthermore, our data support the view that SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion.     

Surfactant Bilayers Maintain Transmembrane Protein Activity

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca²⁺ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase.     

Surfactant Bilayers Maintain Transmembrane Protein Activity

In vitro studies of membrane proteins are of interest only if their structure and function are significantly preserved. One approach is to insert them into the lipid bilayers of highly viscous cubic phases rendering the insertion and manipulation of proteins difficult. Less viscous lipid sponge phases are sometimes used, but their relatively narrow domain of existence can be easily disrupted by protein insertion. We present here a sponge phase consisting of nonionic surfactant bilayers. Its extended domain of existence and its low viscosity allow easy insertion and manipulation of membrane proteins. We show for the first time, to our knowledge, that transmembrane proteins, such as bacteriorhodopsin, sarcoplasmic reticulum Ca²⁺ATPase (SERCA1a), and its associated enzymes, are fully active in a surfactant phase.     

Layilin,A Novel Talin-binding Transmembrane Protein Homologous with C-type Lectins,is Localized in Membrane Ruffles

Changes in cell morphology and motility are mediated by the actin cytoskeleton. Recent advances in our understanding of the regulators of microfilament structure and dynamics have shed light on how these changes are controlled, and efforts continue to define all the structural and signaling components involved in these processes. The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. We report a new binding partner for talin that we have named layilin, which contains homology with C-type lectins, is present in numerous cell lines and tissue extracts, and is expressed on the cell surface. Layilin colocalizes with talin in membrane ruffles, and is recruited to membrane ruffles in cells induced to migrate in in vitro wounding experiments and in peripheral ruffles in spreading cells. A ten–amino acid motif in the layilin cytoplasmic domain is sufficient for talin binding. We have identified a short region within talin''s amino-terminal 435 amino acids capable of binding to layilin in vitro. This region overlaps a binding site for focal adhesion kinase.     

The Novel Membrane Protein TMEM59 Modulates Complex Glycosylation,Cell Surface Expression,and Secretion of the Amyloid Precursor Protein

Ectodomain shedding of the amyloid precursor protein (APP) by the two proteases α- and β-secretase is a key regulatory event in the generation of the Alzheimer disease amyloid β peptide (Aβ). At present, little is known about the cellular mechanisms that control APP shedding and Aβ generation. Here, we identified a novel protein, transmembrane protein 59 (TMEM59), as a new modulator of APP shedding. TMEM59 was found to be a ubiquitously expressed, Golgi-localized protein. TMEM59 transfection inhibited complex N- and O-glycosylation of APP in cultured cells. Additionally, TMEM59 induced APP retention in the Golgi and inhibited Aβ generation as well as APP cleavage by α- and β-secretase cleavage, which occur at the plasma membrane and in the endosomes, respectively. Moreover, TMEM59 inhibited the complex N-glycosylation of the prion protein, suggesting a more general modulation of Golgi glycosylation reactions. Importantly, TMEM59 did not affect the secretion of soluble proteins or the α-secretase like shedding of tumor necrosis factor α, demonstrating that TMEM59 did not disturb the general Golgi function. The phenotype of TMEM59 transfection on APP glycosylation and shedding was similar to the one observed in cells lacking conserved oligomeric Golgi (COG) proteins COG1 and COG2. Both proteins are required for normal localization and activity of Golgi glycosylation enzymes. In summary, this study shows that TMEM59 expression modulates complex N- and O-glycosylation and suggests that TMEM59 affects APP shedding by reducing access of APP to the cellular compartments, where it is normally cleaved by α- and β-secretase.     

Complexin 2 Modulates Vesicle-associated Membrane Protein (VAMP) 2-regulated Zymogen Granule Exocytosis in Pancreatic Acini

Complexins are soluble proteins that regulate the activity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes necessary for vesicle fusion. Neuronal specific complexin 1 has inhibitory and stimulatory effects on exocytosis by clamping trans-SNARE complexes in a prefusion state and promoting conformational changes to facilitate membrane fusion following cell stimulation. Complexins are unable to bind to monomeric SNARE proteins but bind with high affinity to ternary SNARE complexes and with lower affinity to target SNARE complexes. Far less is understood about complexin function outside the nervous system. Pancreatic acini express the complexin 2 isoform by RT-PCR and immunoblotting. Immunofluorescence microscopy revealed complexin 2 localized along the apical plasma membrane consistent with a role in secretion. Accordingly, complexin 2 was found to interact with vesicle-associated membrane protein (VAMP) 2, syntaxins 3 and 4, but not with VAMP 8 or syntaxin 2. Introduction of recombinant complexin 2 into permeabilized acini inhibited Ca²⁺-stimulated secretion in a concentration-dependent manner with a maximal inhibition of nearly 50%. Mutations of the central α-helical domain reduced complexin 2 SNARE binding and concurrently abolished its inhibitory activity. Surprisingly, mutation of arginine 59 to histidine within the central α-helical domain did not alter SNARE binding and moreover, augmented Ca²⁺-stimulated secretion by 130% of control. Consistent with biochemical studies, complexin 2 colocalized with VAMP 2 along the apical plasma membrane following cholecystokinin-8 stimulation. These data demonstrate a functional role for complexin 2 outside the nervous system and indicate that it participates in the Ca²⁺-sensitive regulatory pathway for zymogen granule exocytosis.     

Influenza Hemagglutinin Modulates Phosphatidylinositol 4,5-Bisphosphate Membrane Clustering

The lipid phosphatidylinositol 4,5-bisphosphate (PIP2) forms nanoscopic clusters in cell plasma membranes; however, the processes determining PIP2 mobility and thus its spatial patterns are not fully understood. Using super-resolution imaging of living cells, we find that PIP2 is tightly colocalized with and modulated by overexpression of the influenza viral protein hemagglutinin (HA). Within and near clusters, HA and PIP2 follow a similar spatial dependence, which can be described by an HA-dependent potential gradient; PIP2 molecules move as if they are attracted to the center of clusters by a radial force of 0.079 ± 0.002 pN in HAb2 cells. The measured clustering and dynamics of PIP2 are inconsistent with the unmodified forms of the raft, tether, and fence models. Rather, we found that the spatial PIP2 distributions and how they change in time are explained via a novel, to our knowledge, dynamic mechanism: a radial gradient of PIP2 binding sites that are themselves mobile. This model may be useful for understanding other biological membrane domains whose distributions display gradients in density while maintaining their mobility.