Membrane Binding Ability Research Articles

One-step separation of myristoylated and nonmyristoylated retroviral matrix proteins

N-terminal myristoylation of retroviral matrix proteins is essential for the targeting of the Gag polyproteins to the plasma membrane. To investigate the effect of the myristoylation on the structure and membrane binding ability of the matrix proteins, it is necessary to prepare their myristoylated forms. We present purification of myristoylated matrix proteins of the mouse mammary tumor virus and murine leukemia virus, two morphogenetically distinct retroviruses. The proteins were expressed in Escherichia coli coexpressing a yeast N-myristoyltransferase. This E. coli expression system yielded a mixture of myristoylated and nonmyristoylated matrix proteins. We established efficient one-step metal affinity purification that enabled to obtain pure myristoylated matrix proteins suitable for structural and functional studies.

Probing conformational changes in lipoxygenases upon membrane binding: Fine-tuning by the active site inhibitor ETYA

Lipoxygenases (LOXs) are lipid-peroxidizing enzymes that are involved in the metabolism of polyunsaturated fatty acids. Their biological activity includes a membrane binding process whose molecular details are not completely understood. The mechanism of enzyme–membrane interactions is thought to involve conformational changes at the level of the protein tertiary structure, and the extent of such alterations depends on the degree of structural flexibility of the different LOX isoforms. In this study, we have tested the resilience properties of a plant and a mammalian LOX, by using high pressure fluorescence measurements at different temperatures. The binding of LOXs to the lipid bilayer has been characterized using both large and giant unilamellar vesicles and electron transfer particles (inner mitochondrial membranes) as model membranes. The data indicate that the degree of LOXs' flexibility is strictly dependent on the two distinct N- and C-terminal domains that characterize the 3D structure of these enzymes. Furthermore, they demonstrate that increasing the rigidity of protein scaffolding by the presence of an active site ligand impairs the membrane binding ability of LOXs. These findings provide evidence that the amphitropic nature of LOXs is finely tuned by the interaction of the substrate with the residues of the active site, suggesting new strategies for the design of enzyme inhibitors.

A protein isolated from human oviductal tissue in vitro secretion, identified as human lactoferrin, interacts with spermatozoa and oocytes and modulates gamete interaction

Is lactoferrin (LF) (detected in oviductal secretion) able to bind to oocytes and sperm and modulate gamete interaction? LF binds to zona pellucida (ZP) and spermatozoa (depending upon the capacitation stage and acrosome status) and inhibits gamete interaction in vitro. Proteins from human oviductal tissue secretion modulate gamete interaction and parameters of sperm function in vitro and some of them bind to sperm, but they remain to be isolated and identified. Proteins were isolated from human oviductal tissue secretion using their sperm membrane binding ability. One of the isolated proteins was identified as human LF and immunolocalized in tubal tissues. LF expression was analyzed in native oviductal fluid and oviduct epithelial cells (at different phases of the menstrual cycle: proliferative, periovulatory and secretory). In addition, the LF binding sites on spermatozoa (at different capacitation and acrosome reaction stages) and on ZP and the dose-dependent effect of LF on gamete interaction were investigated. All experiments were performed at least three times. Tubal tissues obtained from premenopausal patients (scheduled for hysterectomy, n = 23) were cultured in DMEM/Ham's F12 medium and conditioned media (CM) were collected. Motile spermatozoa were obtained by swim-up from normozoospermic semen samples from healthy donors (n = 4). An affinity chromatography with sperm membrane extracts was used to isolate proteins from CM. Isolated proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophresis and further identified by nano liquid chromatography tandem mass spectrometry peptide sequencing. The presence of LF in oviductal tissue was investigated by immunohistochemistry and immunofluorescence and was detected in native oviductal fluid and oviduct epithelial cells homogenates by western blot. LF binding sites on gametes were investigated by incubating gametes with the protein coupled to fluorescein isothiocyanate (FITC). The acrosome reaction was assessed with Pisum sativum agglutinin conjugated with rhodamine. The effect of increasing concentrations of LF (0.1-100 µg/ml) on gamete interaction was evaluated by a sperm-ZP binding assay, using human oocytes donated by women undergoing IVF procedures. A protein isolated by the affinity column was identified as human LF. LF was immunolocalized in human oviductal tissue and detected in oviductal fluid and oviduct epithelial cell homogenates. In the latter case, LF expression was highest at the periovulatory phase of the menstrual cycle (P < 0.01). Different LF binding patterns were observed on spermatozoa depending upon capacitation stage and if the acrosome reaction had occurred. Unstained sperm were most prevalent before capacitation, but after incubation for 6 h under capacitating conditions and in acrosome-reacted sperm LF binding was observed, mainly localized in the equatorial segment and post-acrosomal region of the sperm head. LF binding studies on ZP showed homogenous staining. LF caused a dose-dependent significant inhibition of sperm-ZP interaction, and the effect was already significant (P < 0.01) with the lowest LF concentration used. This study has investigated the effect of LF only on human gamete interaction in vitro and thus has some limitations. Further investigations of the potential mechanisms involved in LF action both on gamete function in vitro and in vivo in animal models are needed to confirm the role of this protein in the reproductive process. The present data indicate that human oviductal LF expression is cycle dependent and inhibited gamete interaction in vitro. No previous data were available about potential direct effects of LF on gamete interaction. It could be thought that the protein is involved in the regulation of the reproductive process, perhaps contributing to prevent polyspermy. Thus, further research is needed to clarify the potential role of LF in the regulation of the fertilization process. This study was supported by grants from FONCYT (PICT 01095, S.A.G., M.J.M) and SECyT UNR (PIDBIO238, S.A.G). The authors have no conflict of interest to declare.

A lysophospholipid acyltransferase antagonist, CI-976, creates novel membrane tubules marked by intracellular phospholipase A1 KIAA0725p

CI-976 is a lysophospholipid acyltransferase antagonist that is known to affect secretory and endocytic membrane-trafficking pathways likely by increasing the lysophospholipid content in membranes. Our previous study suggested that lysophospholipids formed through the action of an intracellular phospholipase A(1), KIAA0725p (also known as DDHD2 and iPLA(1)γ), may be important for the association of this enzyme with membranes. In this study, we examined the effect of CI-976 on the membrane association of KIAA0725p. While in HeLa cells KIAA0725p is localized in the Golgi and cytosol, in mouse embryonic fibroblasts (MEFs), it was found to be principally localized in the cytosol with some on post-endoplasmic reticulum compartments including the cis-Golgi. Treatment of MEFs with CI-976 induced the redistribution of KIAA0725p to membrane tubules, which were in vicinity to fragmented mitochondria. These tubules were not decorated with canonical organelle markers including Golgi proteins. A human KIAA0725p mutant, which exhibits decreased membrane-binding ability, was also redistributed to membrane structures upon CI-976 treatment. Our data suggest that the association of KIAA0725p with membranes is regulated by lipid metabolism, and that CI-976 may create unique membrane structures that can be marked by KIAA0725p.

Effects of Sequence Length and Composition on Antimicrobial Peptide Action

Antimicrobial peptides are short, cationic, amphiphilic naturally occurring sequences that have selective antimicrobial properties. The proposed mechanism of action for these peptides is through interaction with and disruption of the bacterial membrane. We have investigated the length and amino acid composition dependence of the antimicrobial peptides ponericin L1 and C18G on antimicrobial activity and membrane binding ability. Truncation of the peptide sequence at different points through synthesis allowed for investigation of length, overall hydrophobicity, and net charge on function. Circular dichroism and fluorescence spectroscopy were used to determine the binding affinity and structure of the peptide in the presence of lipid vesicles. All of the L1 derived peptides exhibited the ability to form alpha helices and bind to the lipid membranes to different degrees. The data suggests that the modified peptide (L1A) and the truncated peptides (L1A-13T, L1A-16T, and L1A-21T) work by forming an alpha helix to permeabilize the bacterial membrane and cause bacteriolysis. Alternatively, the C18G derived truncates exhibited a length threshold in their ability to bind membranes with high affinity and form helical structures. Ongoing experiments are probing membrane topography.

Effect of Lipid Bilayer Composition on the Membrane-Binding Ability and Depth of Insertion of Antimicrobial Piscidin 1: A Solid-State Deuterium NMR Study

Piscidins constitute a family of amphipathic, cationic, antimicrobial peptides (AMPs) found in the skin, gills and gastrointestinal tracts of hybrid striped bass. Unstructured in water, they become alpha-helical in the presence of phospholipids. It is believed that their ability to disrupt phospholipid bilayers is related to their antimicrobial effects on yeasts and bacteria. Interestingly, the minimum inhibitory concentrations of piscidin tend to be higher for yeasts than most bacteria. Here, we used Circular Dichroism (CD) and deuterium solid-state Nuclear Magnetic Resonance (ssNMR) to investigate the membrane-binding ability and depth of insertion of piscidin 1 in different membrane mimetic systems. Aligned lipid bilayer preparations were made using 3:1 POPC/POPG-d31 (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol-d31) and 3:1 POPC/POPS-d31 (1-palmitoyl 2-oleoyl phosphatidylserine-d31) to mimic bacterial and yeast cell membranes, respectively. To test the effects of ergosterol, a sterol found in yeasts, we added 20% ergosterol (molar ratio) to the lipid mixtures. We hypothesized that both PS and ergosterol would reduce the ability of piscidin to bind and disrupt bilayers.Deuterium quadrupolar splittings, which were obtained for deuterons in the methylene and terminal methyl groups of the phospholipid acyl chains, were used to assess the disordering effect of piscidin on the acyl chains. Our results suggest that the type of anionic lipid component (e.g., PG versus PS) does not affect acyl chain disordering by piscidin while ergosterol appears to decrease its disordering effect. The data also give evidence that membrane-bound piscidin is located close to the phospholipid headgroups. Overall, these studies help us gain some insight into specific physicochemical features of lipid membranes that affect piscidin-lipid interactions. The implications in terms of piscidin's mode of action will be discussed.

Structural Studies of Neuropeptide Y and NPY-[18-36] in the Presence of Neuronal Lipid Membrane Mimics

Neuropeptide Y (NPY), a 36-residue long polypeptide and one of the most abundant neuropeptides found in the mammalian nervous system, activates G-protein coupled receptors. As a neurotransmitter, it helps regulate metabolism and the cardiovascular system. It has also been shown to have antimicrobial properties. To better understand multiple functions of NPY, it is important to characterize its structural features under physiologically-relevant conditions. Previous studies have shown that NPY interacts strongly with phospholipids that mimic neuronal cell membranes. In this regard, we recently used CD and solid-state NMR to show that 2:1PC:PS (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/ 1-palmitoyl 2-oleoyl phosphatidylserine), a suitable lipid mimic for neuronal membranes, could be used for high-resolution structural studies of NPY and antagonist NPY-[18-36].In this research, we have used solid-state NMR more extensively on NPY and NPY-[18-36] to investigate their high-resolution structures and dynamics in the presence of 2:1 POPC/POPS bilayers. Two-dimensional heteronuclear correlation solid-state NMR spectra have been collected on 15N-amide backbone labeled peptides in the presence of aligned lipid bilayers. 15N chemical shifts and 15N-1H dipolar couplings have been used to obtain the topological orientation and preliminary backbone structures of the peptides. The data indicate that the alpha-helical portion of the peptides adopt an in-plane bilayer orientation and diffusive rapidly in the plane of the membrane. To investigate the membrane-binding and membrane-disrupting abilities of NPY and NPY-[18-36], we performed CD and dye leakage experiments on samples containing vesicles of various lipid compositions and different peptide concentrations. We will discuss the results in terms of the peptide's ability to disrupt lipid bilayers mimicking mammalian and microbial cells. Overall, these studies provide valuable knowledge to help elucidate the molecular basis of NPY's multiple functions in the human body.

Evidence of a Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Machinery in HIV-1 Assembly and Release

Retrovirus assembly is a complex process that requires the orchestrated participation of viral components and host-cell factors. The concerted movement of different viral proteins to specific sites in the plasma membrane allows for virus particle assembly and ultimately budding and maturation of infectious virions. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins constitute the minimal machinery that catalyzes the fusion of intracellular vesicles with the plasma membrane, thus regulating protein trafficking. Using siRNA and dominant negative approaches we demonstrate here that generalized disruption of the host SNARE machinery results in a significant reduction in human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus particle production. Further analysis of the mechanism involved revealed a defect at the level of HIV-1 Gag localization to the plasma membrane. Our findings demonstrate for the first time a role of SNARE proteins in HIV-1 assembly and release, likely by affecting cellular trafficking pathways required for Gag transport and association with the plasma membrane.

Effects of alkyl chain length of gallate on self-association and membrane binding

Alkyl gallates are anticipated for their use as anti-bacterial and anti-viral agents. Although their pharmacological activities depend on their alkyl chain length, no mechanism has yet been clarified. As described herein, we investigated the membrane binding properties of a series of alkyl gallates using fluorescence measurement to elucidate their different pharmacological activities. Membrane binding of the alkyl gallates increased concomitantly with increasing alkyl chain length, except for cetyl gallate and stearyl gallate. Dynamic light scattering revealed that alkyl gallates with a long alkyl chain are prone to self-association in the solution. Membrane binding abilities of the alkyl gallates are correlated with anti-bacterial and anti-virus activities, as described in previous reports. The partition constants of the alkyl gallates to lipid membranes depend on the membrane components and the membrane phase. Self-association and lipid binding of the alkyl gallates might be primary biophysical factors associated with their pharmacological activities.

19F NMR studies of α‐synuclein‐membrane interactions

Abstractα‐Synuclein function is thought to be related to its membrane binding ability. Solution NMR studies have identified several α‐synuclein‐membrane interaction modes in small unilamellar vesicles (SUVs), but how membrane properties affect binding remains unclear. Here, we use 19F NMR to study α‐synuclein‐membrane interactions by using 3‐fluoro‐L‐tyrosine (3FY) and trifluoromethyl‐L‐phenylalanine (tfmF) labeled proteins. Our results indicate that the affinity is affected by both the head group and the acyl chain of the SUV. Negatively charged head groups have higher affinity, but different head groups with the same charge also affect binding. We show that the saturation of the acyl chain has a dramatic effect on the α‐synuclein‐membrane interactions by studying lipids with the same head group but different chains. Taken together, the data show that α‐synuclein's N‐terminal region is the most important determinate of SUV binding, but its C‐terminal region also modulates the interactions. Our data support the existence of multiple tight phospholipid‐binding modes, a result incompatible with the model that α‐synuclein lies solely on the membrane surface.

Dissecting BAR domain function in the yeast Amphiphysins Rvs161 and Rvs167 during endocytosis.

BAR domains are protein modules that bind to membranes and promote membrane curvature. One type of BAR domain, the N-BAR domain, contains an additional N-terminal amphipathic helix, which contributes to membrane-binding and bending activities. The only known N-BAR-domain proteins in the budding yeast Saccharomyces cerevisiae, Rvs161 and Rvs167, are required for endocytosis. We have explored the mechanism of N-BAR-domain function in the endocytosis process using a combined biochemical and genetic approach. We show that the purified Rvs161-Rvs167 complex binds to liposomes in a curvature-independent manner and promotes tubule formation in vitro. Consistent with the known role of BAR domain polymerization in membrane bending, we found that Rvs167 BAR domains interact with each other at cortical actin patches in vivo. To characterize N-BAR-domain function in endocytosis, we constructed yeast strains harboring changes in conserved residues in the Rvs161 and Rvs167 N-BAR domains. In vivo analysis of the rvs endocytosis mutants suggests that Rvs proteins are initially recruited to sites of endocytosis through their membrane-binding ability. We show that inappropriate regulation of complex sphingolipid and phosphoinositide levels in the membrane can impinge on Rvs function, highlighting the relationship between membrane components and N-BAR-domain proteins in vivo.

A nuclear ligand MRG15 involved in the proapoptotic activity of medicinal fungal galectin AAL ( Agrocybe aegerita lectin)

Background We have previously reported a novel fungal galectin Agrocybe aegerita lectin (AAL) with apoptosis-induced activity and nuclear migration activity. The importance of nuclear localization for AAL's apoptosis-induced activity has been established by mutant study. However, the mechanism remains unclear. Methods We further investigated the mechanism using a previously reported carbohydrate recognition domain (CRD) mutant protein H59Q, which retained its nuclear localization activity but lost most of its apoptotic activity. The cell membrane-binding ability of recombinant AAL (rAAL) and H59Q was analyzed by FACS, and their cellular partners were identified by affinity chromatography and mass spectroscopy. Furthermore, the interaction of AAL and ligand was proved by mammalian two-hybrid and pull down assays. A knockdown assay was used to confirm the role of the ligand. Results The apoptotic activity of AAL could be blocked by lactose. Mutant H59Q retained comparable cell membrane-binding ability to rAAL. Four cellular binding partners of AAL in HeLa cells were identified: glucose-regulated protein 78 (GRP78); mortality factor 4-like protein 1 (MRG15); elongation factor 2 (EEF2); and heat shock protein 70 (Hsp70). CRD region of AAL was required for the interaction between AAL/mutant AAL and MRG15. MRG15 knockdown increased the cells' resistance to AAL treatment. Conclusion MRG15 was a nuclear ligand for AAL in HeLa cells. These data implied the existence of a novel nuclear pathway for the antitumor activity of fungal galectin AAL. General significance These findings provide a novel explanation of AAL bioactivity and contribute to the understanding of mushroom lectins' antitumor activity.

A novel role for iron in modulating the activity and membrane‐binding ability of a trimmed soybean lipoxygenase‐1

Lipoxygenases (LOXs) are iron-containing enzymes that play critical roles in plants and animals. As yet, metal atom extraction, reconstitution, and substitution have not been successfully applied to soybean LOX-1 [Glycine max (L.) Merrill], a prototype member of the LOX family that is widely used in structural and kinetic studies. Here, tryptic digestion of native LOX-1, used as a control, allowed us to isolate the 60-kDa C-terminal region (termed miniLOX), that retains the catalytically active iron in a more accessible position. Then, iron was removed to obtain an unprecedented apo-miniLOX, which was reconstituted and substituted with different metal ions. These forms of miniLOX were characterized vs. native LOX-1 by kinetic analysis, near UV circular dichroism, steady-state fluorescence, and fluorescence resonance energy transfer. MiniLOX showed a 2-fold increase in the membrane-binding affinity compared with native LOX-1 and a remarkable 4-fold increase compared with apo-miniLOX (K(d)=9.2+/-1.0, 17.9+/-2.0, and 45.4+/-4.3 microM, respectively). Furthermore, miniLOX reconstituted with Fe(II) or Fe(III) partially recovered its membrane-binding ability (K(d)=21.4+/-2.4 and 18.9+/-5.5 microM, respectively), overall supporting a novel noncatalytic role for iron in the LOX family.

Fluorescence Polarization Analysis for Revealing Molecular Mechanism of Nucleotide-Dependent Phospholipid Membrane Binding of MinD Adenosine 5'-Triphosphate, Adenosine Triphosphatase

Membrane binding of Walker type adenosine 5'-triphosphate, adenosine triphosphatase (ATPase), MinD, is a key step in regulating the site of cell division in Escherichia coli. Two lysine residues (K11, K16) in the Walker A motif of MinD have been suggested to be essential for both membrane binding and ATPase activity, but the relationship between the membrane binding of MinD and its ATPase activity is still unclear. To reveal the role of K11 and K16 in MinD membrane interaction and ATP-binding, we compared the functionality of wild-type MinD (WT) and two MinD mutants that lack ATPase activity, where alanine was substituted for lysine at positions 11 and 16 (K11A, K16A), using liposomes and fluorescent-labeled ATP. The ATP dissociation constant (K(d)) of wild-type MinD was 4.9 µM. Unexpectedly, the K(d) values of the two lysine mutants were almost the same as that of wild type, indicating that ATP can bind to MinD mutants, even though these mutants showed no ATPase activity and membrane binding ability. Our results presumed that K11 and K16 residues might play an important role in dimmer formation of MinD, but not ATP binding step, for recruiting to membrane.

Disulfide bond as a structural determinant of prion protein membrane insertion.

Conversion of the normal soluble form of prion protein, PrP (PrP(C)), to proteinase K-resistant form (PrP(Sc)) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from alpha-helix to beta-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

Development of lateral flow membranes for immunoassay separation

The lateral flow nitrocellulose membrane is one of the commonly used separation media for bacteria detection in drinking water treatment facilities. In order to enhance its performance, control of the membrane surface and cross-section morphology is primarily important. The challenge of this study is to combine various formulations and casting variables to obtain lateral flow nitrocellulose membranes with the desired morphologies. Through the dry-phase inversion method, the drying temperature was found to be an important parameter in synthesizing membranes as it affects pore structures. A high drying temperature causes agglomeration of the polymer matrix, and thus smaller pores were observed on the membrane surface. This further decreases the membrane lateral liquid migration rate, besides reducing the membrane binding ability for bacteria detection. Results show that by decreasing polymer concentration, membrane surface pores became apparently larger, thus creating a faster lateral migration speed of the water solution. A larger pore size increases the chance for the bacteria detecting agent to bind onto the pore layers, which ultimately enhances the bacteria detection ability of the device.

Structural Properties of Plant and Mammalian Lipoxygenases. Temperature-Dependent Conformational Alterations and Membrane Binding Ability

Lipoxygenases form a heterogeneous family of lipid peroxidizing enzymes, which have been implicated in the synthesis of inflammatory mediators, in cell development and in the pathogenesis of various diseases with major health and political relevance (atherosclerosis, osteoporosis). The crystal structures of various lipoxygenase-isoforms have been reported, and X-ray coordinates for enzyme-ligand complexes are also available. Although the 3D-structures of plant and animal lipoxygenase-isoforms are very similar, recent small-angle X-ray scattering data suggested a higher degree of motional flexibility of mammalian isozymes in aqueous solutions. To explore the molecular basis for these differences we performed dynamic fluorescence measurements that allowed us to study temperature-induced conformational changes arising from three-dimensional fluctuations of the protein matrix. For this purpose, we first investigated the impact of elevated temperature on activity, secondary structure, tertiary structure dynamics and conformational alterations. Applying fluorescence resonance energy transfer we also tested the membrane binding properties of the two lipoxygenase-isoforms, and compared their binding parameters. Taken together, our results indicate that the rabbit 12/15-lipoxygenase is more susceptible to temperature-induced structural alterations than the soybean enzyme. Moreover, the rabbit enzyme exhibits a higher degree of conformational flexibility of the entire protein molecule (global flexibility) and offers the possibility of augmented substrate movement at the catalytic center (local flexibility).

Defect of Human Immunodeficiency Virus Type 2 Gag Assembly in Saccharomyces cerevisiae

We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.

Incorporation of Human Immunodeficiency Virus Type 1 Reverse Transcriptase into Virus-Like Particles

We demonstrate that a genetically engineered human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) composed mainly of p66 or p51 subunits can be incorporated into virus-like particles (VLPs) when coexpressed with HIV-1 Pr55(gag). VLP-associated RT exhibited a detergent-resistant association with immature cores during sucrose gradient equilibrium centrifugation, suggesting that RT is incorporated into VLPs. However, RT that retains downstream integrase (IN) is severely inhibited in terms of incorporation into VLPs. Results from immunofluorescence tests reveal that RT-IN is primarily localized at the perinuclear area and exhibits poor colocalization with Gag. IN removal leads to a redistribution of RT throughout the cytoplasm and improved RT incorporation into VLPs. Similar results were observed for RT-IN in which alanine was substituted for 186-Lys-Arg-Lys-188 residues of the IN putative nuclear localization signal, suggesting that IN karyophilic properties may partly account for the inhibitory effect of IN on RT incorporation. Although the membrane-binding capacity of RT was markedly reduced compared to that of wild-type Gag or Gag-Pol, the correlation of membrane-binding ability with particle incorporation efficiency was incomplete. Furthermore, we observed that membrane-binding-defective myristylation-minus RT can be packaged into VLPs at the same level as its normal myristylated counterpart. This suggests that the incorporation of RT into VLPs is independent of membrane affinity but very dependent on RT-Gag interaction. Results from a genetic analysis suggest that the Gag-interacting regions of RT mainly reside in the thumb subdomain and that the RT-binding domains of Gag are located in the matrix (MA) and p6 regions.

The RAC Binding Domain/IRSp53-MIM Homology Domain of IRSp53 Induces RAC-dependent Membrane Deformation

The concave surface of the crescent-shaped Bin-amphiphysin-Rvs (BAR) domain is postulated to bind to the cell membrane to induce membrane deformation of a specific curvature. The Rac binding (RCB) domain/IRSp53-MIM homology domain (IMD) has a dimeric structure that is similar to the structure of the BAR domain; however, the RCB domain/IMD has a "zeppelin-shaped" dimer. Interestingly, the RCB domain/IMD of IRSp53 possesses Rac binding, membrane binding, and actin filament binding abilities. Here we report that the RCB domain/IMD of IRSp53 induces membrane deformation independent of the actin filaments in a Rac-dependent manner. In contrast to the BAR domain, the RCB domain/IMD did not cause long tubulation of the artificial liposomes; however, the Rac binding domain caused the formation of small buds on the liposomal surface. When expressed in cells, the Rac binding domain induced outward protrusion of the plasma membrane in a direction opposite to that induced by the BAR domain. Mapping of the amino acids responsible for membrane deformation suggests that the convex surface of the Rac binding domain binds to the membrane in a Rac-dependent manner, which may explain the mechanism of the membrane deformation induced by the RCB domain/IMD.