Myristoylation of human LanC-like Protein 2 (LANCL2) is essential for the interaction with the plasma membrane and the increase in cellular sensitivity to adriamycin

Human LANCL2, also known as Testis-specific Adriamycin Sensitivity Protein (TASP), is a member of the highly conserved and widely distributed lanthionine synthetase component C-like (LANCL) protein family. Expression studies of tagged LANCL2 revealed the major localization to the plasma membrane, juxta-nuclear vesicles, and the nucleus, in contrast to the homologue LANCL1 that was mainly found in the cytosol and nucleus. We identified the unique N-terminus of LANCL2 to function as the membrane anchor and characterized the relevant N-terminal myristoylation and a basic phosphatidylinositol phosphate-binding site. Interestingly, the non-myristoylated protein was confined to the nucleus indicating that the myristoylation targets LANCL2 to the plasma membrane. Cholesterol depletion by methyl-β-cyclodextrin caused the partial dissociation of overexpressed LANCL2 from the plasma membrane in vitro, whereas in vivo we observed an enhanced cell detachment from the matrix. We found that overexpressed LANCL2 interacts with the cortical actin cytoskeleton and therefore may play a role in cytoskeleton reorganization and in consequence to cell detachment. Moreover, we confirmed previous data that LANCL2 overexpression enhances the cellular sensitivity to the anticancer drug adriamycin and found that this sensitivity is dependent on the myristoylation and membrane association of LANCL2.     

Myristoylation of the RING finger Z protein is essential for arenavirus budding

The arenavirus small RING finger Z protein is the main driving force of arenavirus budding. The primary structure of Z is devoid of hydrophobic transmembrane domains, but both lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus Z proteins accumulate near the inner surface of the plasma membrane and are strongly membrane associated. All known arenavirus Z proteins contain a glycine (G) at position 2, which is a potential acceptor site for a myristoyl moiety. Metabolic labeling showed incorporation of [(3)H]myristic acid by wild-type Z protein but not by the G2A mutant. The mutation G2A eliminated Z-mediated budding. Likewise, treatment with the myristoylation inhibitor 2-hydroxymyristic acid inhibited Z-mediated budding, eliminated formation of virus-like particles, and caused a dramatic reduction in virus production in LCMV-infected cells. Budding activity was restored in G2A mutant Z proteins by the addition of the myristoylation domain of the tyrosine protein kinase Src to their N termini. These findings indicate N-terminal myristoylation of Z plays a key role in arenavirus budding.     

The Ca2+- and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, H_II). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.     

Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement

Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.     

Diferric transferrin reductase in the plasma membrane is inhibited by adriamycin

Diferric transferrin which is often necessary for growth of cells is reduced by the transplasma membrane electron transport system of HeLa cells with release of ferrous iron outside the cell. Reduction of external diferric transferrin is reflected in oxidation of internal NADH. Adriamycin, an antitumor drug, inhibits diferric transferrin reduction by the HeLa cells and inhibits concomittant oxidation of cytosolic NADH at concentrations, 10(-8)-10(-6)M, which inhibit cell growth. Isolated liver plasma membranes have an NADH diferric transferrin reductase activity which is inhibited by similar adriamycin concentrations. We propose that inhibition of cell growth by adriamycin can be based on inhibition of transplasmalemma diferric transferrin reductase.     

Rab11b is essential for recycling of transferrin to the plasma membrane

Members of the Rab family of small GTPases play important roles in membrane trafficking along the exocytic and endocytic pathways. The Rab11 subfamily consists of two highly conserved members, Rab11a and Rab11b. Rab11a has been localized both to the pericentriolar recycling endosome and to the trans-Golgi network and functions in recycling of transferrin. However, the localization and function of Rab11b are completely unknown. In this study green fluorescent protein (GFP)-tagged Rab11b was used to determine its subcellular localization. GFP-Rab11b colocalized with internalized transferrin, and using different mutants of Rab11b, the role of this protein in transferrin uptake and recycling was examined. Two of these mutants, Rab11b-Q/L (constitutively active) and Rab11b-S/N (constitutively inactive), strongly inhibited the recycling of transferrin. Interestingly, both of them had no effect on transferrin uptake. In contrast, the C-terminally altered mutant Rab11b-DeltaC, which cannot be prenylated and therefore cannot interact with membranes, did not interfere with wild-type Rab11b function. From these data we concluded that functional Rab11b is essential for the transport of internalized transferrin from the recycling compartment to the plasma membrane.     

Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane

Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation.     

Myristoylation of human immunodeficiency virus type 1 gag protein is required for efficient env protein transportation to the surface of cells

Highly conserved amino acids in the N-terminal region of the human immunodeficiency virus type 1 (HIV-1) Pr55(gag) are recognized to be critical for the attachment of myristic acid. We previously reported that the env protein was not detected on the cell surface by blocking of N-myristoylation of Pr55(gag) with N-myristoyl glycinal diethylacetal. Here, we constructed a mutant by substituting the N-terminal glycine of Pr55(gag) with alanine to demonstrate that N-myristoylation of Pr55(gag) is required for efficient env protein transportation to the cell surface. The expression level of the env protein on the surface of Jurkat cells transfected with the myristoylation-defective phenotype was observed to be significantly reduced by electron microscopic analyses with a gold-labeled monoclonal antibody against the env protein. In addition, Jurkat cells transfected with the myristoylation-defective phenotype lost the ability of envelope-mediated cell-to-cell fusion. The results suggest that N-myristoylation of the HIV-1 gag protein is necessary for efficient env protein transportation to the cell surface.     

Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins

Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types.     

Different sensitivity to trypsin of the human platelet plasma and intracellular membrane Ca2+ pumps

The Ca2+ pumps associated with human platelet plasma and intracellular membranes have been further characterized by their sensitivity to trypsin. (a) Tryptic degradation of the Ca2+-ATPases has been followed by immunoblotting. It resulted in fragmentation into peptides of 80, 55, 35, and 24 kDa for both enzymes. Subcomplete hydrolysis obtained with a ratio of trypsin/membrane protein of 0.05-0.1 for the two Ca2+ pumps resulted in the total disappearance of the 100-, 80-, and 35-kDa fragments. However, maximum degradation was reached within 1 min for the intracellular enzyme but needed 5 min of incubation for the plasma membrane enzyme. (b) This effect of trypsin has been correlated with its effect on both the Ca2+-ATPase activities. The plasma membrane enzyme showed a maximum inhibition of 50-60% which was obtained using a trypsin/protein ratio of 0.1 and 5 min of incubation. A much higher trypsin sensitivity was observed for the intracellular enzyme because the maximum inhibition reached 80% after only 1 min of incubation. (c) Finally, the two Ca2+ transport systems studied showed different trypsin reactivities; the Ca2+ uptake by the plasma membrane vesicles was inhibited by 20-25%, and this maximum inhibition was observed after 5 min of incubation with trypsin. In contrast, the Ca2+ transport associated with the intracellular membrane vesicles was difficult to detect after trypsin treatment. Taken together, the results show that the two Ca2+ pumps can be distinguished by their trypsin sensitivity.     

Identification of functional domains in sarcoglycans essential for their interaction and plasma membrane targeting

Mutations in sarcoglycans have been reported to cause autosomal-recessive limb-girdle muscular dystrophies. In skeletal and cardiac muscle, sarcoglycans are assembled into a complex on the sarcolemma from four subunits (alpha, beta, gamma, delta). In this report, we present a detailed structural analysis of sarcoglycans using deletion study, limited proteolysis and co-immunoprecipitation. Our results indicate that the extracellular regions of sarcoglycans consist of distinctive functional domains connected by proteinase K-sensitive sites. The N-terminal half domains are required for sarcoglycan interaction. The C-terminal half domains of beta-, gamma- and delta-sarcoglycan consist of a cysteine-rich motif and a previously unrecognized conserved sequence, both of which are essential for plasma membrane localization. Using a heterologous expression system, we demonstrate that missense sarcoglycan mutations affect sarcoglycan complex assembly and/or localization to the cell surface. Our data suggest that the formation of a stable complex is necessary but not sufficient for plasma membrane targeting. Finally, we provide evidence that the beta/delta-sarcoglycan core can associate with the C-terminus of dystrophin. Our results therefore generate important information on the structure of the sarcoglycan complex and the molecular mechanisms underlying the effects of various sarcoglycan mutations in muscular dystrophies.     

The dynamin-like protein ADL1C is essential for plasma membrane maintenance during pollen maturation

Dynamin-related GTPases regulate a wide variety of dynamic membrane processes in eukaryotes. Here, we investigated the function of ADL1C, a member of the Arabidopsis 68 kDa dynamin-like protein family. Analysis of heterozygous adl1C-1 indicates that the mutation specifically affects post-meiotic male gametogenesis. Fifty percent of the mature pollen from heterozygous adl1C-1 androecia are shriveled and fail to germinate in vitro. During microspore maturation, adl1C-1 pollen grains display defects in the plasma membrane and intine morphology, suggesting that ADL1C is essential for the formation and maintenance of the pollen cell surface and viability during desiccation. Consistent with a role in cell-surface dynamics, immunofluorescence microscopy indicates that ADL1C is localized to the cell plate of dividing somatic cells and to the tip of expanding root hairs. We propose that ADL1C functions in plasma membrane dynamics, and we discuss the role of the ADL1 family in plant growth and development.     

Regulatory interaction of the Galpha protein with phospholipase A2 in the plasma membrane of Eschscholzia californica

Plant heterotrimeric G-proteins are involved in a variety of signaling pathways, though only one alpha and a few betagamma isoforms of their subunits exist. In isolated plasma membranes of California poppy (Eschscholzia californica), the plant-specific Galpha subunit was isolated and identified immunologically and by homology of the cloned gene with that of several plants. In the same membrane, phospholipase A(2) (PLA(2)) was activated by yeast elicitor only if GTPgammaS (an activator of Galpha) was present. From the cholate-solubilized membrane proteins, PLA(2) was co-precipitated together with Galpha by a polyclonal antiserum raised against the recombinant Galpha. In this immunoprecipitate and in the plasma membrane (but not in the Galpha-free supernatant) PLA(2) was stimulated by GTPgammaS. Plasma membranes and immunoprecipitates obtained from antisense transformants with a low Galpha content allowed no such stimulation. An antiserum raised against the C-terminus (which in animal Galphas is located near the target coupling site) precipitated Galpha without any PLA(2) activity. Using non-denaturing PAGE, complexes of solubilized plasma membrane proteins were visualized that contained Galpha plus PLA(2) activity and dissociated at pH 9.5. At this pH, PLA(2) was no longer stimulated by GTPgammaS. It is concluded that a distinct fraction of the plasma membrane-bound PLA(2) exists in a detergent-resistant complex with Galpha that can be dissociated at pH 9.5. This complex allows the Galpha-mediated activation of PLA(2).     

The simultaneous production of phosphatidic acid and diacylglycerol is essential for the translocation of protein kinase Cepsilon to the plasma membrane in RBL-2H3 cells

To evaluate the role of the C2 domain in protein kinase Cepsilon (PKCepsilon) localization and activation after stimulation of the IgE receptor in RBL-2H3 cells, we used a series of mutants located in the phospholipid binding region of the enzyme. The results obtained suggest that the interaction of the C2 domain with the phospholipids in the plasma membrane is essential for anchoring the enzyme in this cellular compartment. Furthermore, the use of specific inhibitors of the different pathways that generate both diacylglycerol and phosphatidic acid has shown that the phosphatidic acid generated via phospholipase D (PLD)-dependent pathway, in addition to the diacylglycerol generated via phosphoinosite-phospholipase C (PLC), are involved in the localization of PKCepsilon in the plasma membrane. Direct stimulation of RBL-2H3 cells with very low concentrations of permeable phosphatidic acid and diacylglycerol exerted a synergistic effect on the plasma membrane localization of PKCepsilon. Moreover, the in vitro kinase assays showed that both phosphatidic acid and diacylglycerol are essential for enzyme activation. Together, these results demonstrate that phosphatidic acid is an important and essential activator of PKCepsilon through the C2 domain and locate this isoenzyme in a new scenario where it acts as a downstream target of PLD.     

An interaction between insulin-like growth factor-binding protein 2 (IGFBP2) and integrin alpha5 is essential for IGFBP2-induced cell mobility

In the study we report here, we tested the hypothesis that insulin-like growth factor-binding protein 2 (IGFBP2) promotes cell mobility through its interaction with integrin alpha5. Our previous microarray studies showed that IGFBP2 activates the expression of integrin alpha5. In addition, IGFBP2 has an Arg-Gly-Asp (RGD) domain, which is a known integrin binding motif. We first confirmed our microarray results by showing that the expression of integrin alpha5 is indeed up-regulated at the protein level in IGFBP2-overexpressing SNB19 glioma cells. Using co-immunoprecipitation, we confirmed that IGFBP2 does interact with integrin alpha5. To confirm that IGFBP2 interacts directly with integrin alpha5 through the RGD domain, we created an RGD --> RGE mutant (D306E) IGFBP2 and stably overexpressed the mutant IGFBP2 in the same cell line. Co-immunoprecipitation then showed that D306E-IGFBP2 had no detectable binding with integrin alpha5. We further observed that IGFBP2-overexpressing cells have extensive cell surface lamellipodia, whereas D306E-IGFBP2-overexpressing cells show abundant cell surface focal adhesions. Consistent with this, phenotype analysis then showed that IGFBP2-overexpressing cells have elevated migration rates compared with vector control; in contrast, the migration rates of the D306E-IGFBP2-overexpressing cells were not elevated and were comparable with that of vector control. Decreased expression of integrin alpha5 by small interference RNA in IGFBP2-overexpressing cells also reduced cell mobility. Therefore, we have concluded that one mechanism by which IGFBP2 activates IGFBP2-induced cell mobility is through its interaction with integrin alpha5 and this interaction is specifically mediated through the RGD domain on IGFBP2.     

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection

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No primexine and plasma membrane undulation is essential for primexine deposition and plasma membrane undulation during microsporogenesis in Arabidopsis

Primexine deposition and plasma membrane undulation are the initial steps of pollen wall formation. However, little is known about the genes involved in this important biological process. Here, we report a novel gene, NO PRIMEXINE AND PLASMA MEMBRANE UNDULATION (NPU), which functions in the early stage of pollen wall development in Arabidopsis (Arabidopsis thaliana). Loss of NPU function causes male sterility due to a defect in callose synthesis and sporopollenin deposition, resulting in disrupted pollen in npu mutants. Transmission electronic microscopy observation demonstrated that primexine deposition and plasma membrane undulation are completely absent in the npu mutants. NPU encodes a membrane protein with two transmembrane domains and one intracellular domain. In situ hybridization analysis revealed that NPU is strongly expressed in microspores and the tapetum during the tetrad stage. All these results together indicate that NPU plays a vital role in primexine deposition and plasma membrane undulation during early pollen wall development.     

The N-glycan of the SCR 2 region is essential for membrane cofactor protein (CD46) to function as a measles virus receptor.

Membrane cofactor protein (MCP) (CD46), a complement-regulatory protein, serves as a cellular receptor for measles virus. Its amino-terminal portion is composed of four short consensus repeats (SCR), three of which (SCR1, SCR2, and SCR4) carry an N-linked oligosaccharide. In order to determine the importance of the three N-glycans for the function of MCP as a measles virus receptor, we established Chinese hamster ovary (CHO) cell lines that stably express mutant MCPs lacking one of the three motifs for N glycosylation (NQ1, NQ2, and NQ4). In an additional mutant (NQ1-2), two glycosylation motifs were altered, allowing the addition of an N-linked oligosaccharide only in SCR4. The abilities of the mutant MCPs to function as measles virus receptors were analyzed with three different assays: (i) binding of measles virus hemagglutinin to MCP immobilized on nitrocellulose; (ii) binding of measles virus to CHO cells expressing wild-type or mutant MCP; and (iii) infection of the transfected CHO cells by measles virus. In all three assays, the abilities of the NQ2 and NQ1-2 mutants to serve as measles virus receptors were drastically impaired. The NQ1 and NQ4 mutants were recognized by measles virus almost as efficiently as the wild-type protein. These results indicate that the N-glycan attached to SCR2 is essential for MCP to serve as a measles virus receptor, while the oligosaccharides attached to SCR1 and SCR4 are of only minor importance.     

Reciprocal interaction with G-actin and tropomyosin is essential for aquaporin-2 trafficking

Trafficking of water channel aquaporin-2 (AQP2) to the apical membrane and its vasopressin and protein kinase A (PKA)-dependent regulation in renal collecting ducts is critical for body water homeostasis. We previously identified an AQP2 binding protein complex including actin and tropomyosin-5b (TM5b). We show that dynamic interactions between AQP2 and the actin cytoskeleton are critical for initiating AQP2 apical targeting. Specific binding of AQP2 to G-actin in reconstituted liposomes is negatively regulated by PKA phosphorylation. Dual color fluorescence cross-correlation spectroscopy reveals local AQP2 interaction with G-actin in live epithelial cells at single-molecule resolution. Cyclic adenosine monophosphate signaling and AQP2 phosphorylation release AQP2 from G-actin. In turn, AQP2 phosphorylation increases its affinity to TM5b, resulting in reduction of TM5b bound to F-actin, subsequently inducing F-actin destabilization. RNA interference-mediated knockdown and overexpression of TM5b confirm its inhibitory role in apical trafficking of AQP2. These findings indicate a novel mechanism of channel protein trafficking, in which the channel protein itself critically regulates local actin reorganization to initiate its movement.