Regulating Membrane Association Research Articles

Molecular basis of product recognition during PIP5K-mediated production of PI(4,5)P2 with positive feedback

The ability for cells to localize and activate peripheral membrane-binding proteins is critical for signal transduction. Ubiquitously important in these signaling processes are phosphatidylinositol phosphate (PIP) lipids, which are dynamically phosphorylated by PIP lipid kinases on intracellular membranes. Functioning primarily at the plasma membrane, phosphatidylinositol-4-phosphate 5-kinases (PIP5K) catalyzes the phosphorylation of PI(4)P to generate most of the PI(4,5)P2 lipids found in eukaryotic plasma membranes. Recently, we determined that PIP5K displays a positive feedback loop based on membrane-mediated dimerization and cooperative binding to its product, PI(4,5)P2. Here, we examine how two motifs contribute to PI(4,5)P2 recognition to control membrane association and catalysis of PIP5K. Using a combination of single molecule TIRF microscopy and kinetic analysis of PI(4)P lipid phosphorylation, we map the sequence of steps that allow PIP5K to cooperatively engage PI(4,5)P2. We find that the specificity loop regulates the rate of PIP5K membrane association and helps orient the kinase to more effectively bind PI(4,5)P2 lipids. After correctly orienting on the membrane, PIP5K transitions to binding PI(4,5)P2 lipids near the active site through a motif previously referred to as the substrate or PIP-binding motif (PIPBM). The PIPBM has broad specificity for anionic lipids and serves a role in regulating membrane association invitro and invivo. Overall, our data supports a two-step membrane-binding model where the specificity loop and PIPBM act in concert to help PIP5K orient and productively engage anionic lipids to drive the positive feedback during PI(4,5)P2 production.

Clustered Lysine Residues of the Canine Distemper Virus Matrix Protein Regulate Membrane Association and Budding Activity.

The canine distemper virus (CDV) matrix (M) protein is multifunctional; it orchestrates viral assembly and budding, drives the formation of virus-like particles (VLPs), regulates viral RNA synthesis, and may support additional functions. CDV M may assemble into dimers, where each protomer is constituted by N-terminal and C-terminal domains (NTD and CTD, respectively). Here, to investigate whether electrostatic interactions between CDV M and the plasma membrane (PM) may contribute to budding activity, selected surface-exposed positively charged lysine residues, which are located within a large basic patch of CTD, were replaced by amino acids with selected properties. We found that some M mutants harboring amino acids with neutral and positive charge (methionine and arginine, respectively) maintained full functionality, including proper interaction and localization with the PM as well as intact VLP and progeny virus production as demonstrated by employing a cell exit-complementation system. Conversely, while the overall structural integrity remained mostly unaltered, most of the nonconservative M variants (carrying a glutamic acid; negatively charged) exhibited a cytosolic phenotype secondary to the lack of interaction with the PM. Consequently, such M variants were entirely defective in VLP production and viral particle formation. Furthermore, the proteasome inhibitor bortezomib significantly reduced wild-type M-mediated VLP production. Nevertheless, in the absence of the compound, all engineered M lysine variants exhibited unaffected ubiquitination profiles, consistent with other residues likely involved in this functionally essential posttranslational modification. Altogether, our data identified multiple surface-exposed lysine residues located within a basic patch of CDV M-CTD, critically contributing to PM association and ensuing membrane budding activity.IMPORTANCE Although vaccines against some morbilliviruses exist, infections still occur, which can result in dramatic brain disease or fatal outcome. Postexposure prophylaxis with antivirals would support global vaccination campaigns. Unfortunately, there is no efficient antiviral drug currently approved. The matrix (M) protein of morbilliviruses coordinates viral assembly and egress through interaction with multiple cellular and viral components. However, molecular mechanisms supporting these functions remain poorly understood, which preclude the rationale design of inhibitors. Here, to investigate potential interactions between canine distemper virus (CDV) M and the plasma membrane (PM), we combined structure-guided mutagenesis of selected surface-exposed lysine residues with biochemical, cellular, and virological assays. We identified several lysines clustering in a basic patch microdomain of the CDV M C-terminal domain, which contributed to PM association and budding activity. Our findings provide novel mechanistic information of how morbilliviruses assemble and egress from infected cells, thereby delivering bases for future antiviral drug development.

Autophagy-Related Protein ATG18 Regulates Apicoplast Biogenesis in Apicomplexan Parasites.

ABSTRACTMechanisms by which 3′-phosphorylated phosphoinositides (3′-PIPs) regulate the development of apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii are poorly understood. The catabolic process of autophagy, which is dependent on autophagy-related proteins (ATGs), is one of the major targets of 3′-PIPs in yeast and mammals. In the present study, we identified autophagy-related protein ATG18 as an effector of 3′-PIPs in these parasites. P. falciparum ATG18 (PfATG18) and T. gondii ATG18 (TgATG18) interact with 3′-PIPs but exhibited differences in their specificity of interaction with the ligand PIP. The conditional knockdown of T. gondii or P. falciparum ATG18 (Tg/PfATG18) impaired replication of parasites and resulted in their delayed death. Intriguingly, ATG18 depletion resulted in the loss of the apicomplexan parasite-specific nonphotosynthetic plastid-like organelle apicoplast, which harbors the machinery for biosynthesis of key metabolites, and the interaction of ATG18 to phosphatidylinositol 3-phosphate (PI3P) was critical for apicoplast inheritance. Furthermore, ATG18 regulates membrane association and apicoplast localization of ATG8. These findings provide insights into a novel noncanonical role of ATG18 in apicoplast inheritance. This function of ATG18 in organelle biogenesis is unprecedented in any organism and may be conserved across most apicomplexan parasites.

PROTEIN S-ACYL TRANSFERASE10 Is Critical for Development and Salt Tolerance in Arabidopsis

Protein S-acylation, commonly known as palmitoylation, is a reversible posttranslational modification that catalyzes the addition of a saturated lipid group, often palmitate, to the sulfhydryl group of a Cys. Palmitoylation regulates enzyme activity, protein stability, subcellular localization, and intracellular sorting. Many plant proteins are palmitoylated. However, little is known about protein S-acyl transferases (PATs), which catalyze palmitoylation. Here, we report that the tonoplast-localized PAT10 is critical for development and salt tolerance in Arabidopsis thaliana. PAT10 loss of function resulted in pleiotropic growth defects, including smaller leaves, dwarfism, and sterility. In addition, pat10 mutants are hypersensitive to salt stresses. We further show that PAT10 regulates the tonoplast localization of several calcineurin B-like proteins (CBLs), including CBL2, CBL3, and CBL6, whose membrane association also depends on palmitoylation. Introducing a C192S mutation within the highly conserved catalytic motif of PAT10 failed to complement pat10 mutants, indicating that PAT10 functions through protein palmitoylation. We propose that PAT10-mediated palmitoylation is critical for vacuolar function by regulating membrane association or the activities of tonoplast proteins.

Genomics and Localization of the Arabidopsis DHHC-Cysteine-Rich Domain S-Acyltransferase Protein Family

Protein lipid modification of cysteine residues, referred to as S-palmitoylation or S-acylation, is an important secondary and reversible modification that regulates membrane association, trafficking, and function of target proteins. This enzymatic reaction is mediated by protein S-acyl transferases (PATs). Here, the phylogeny, genomic organization, protein topology, expression, and localization pattern of the 24 PAT family members from Arabidopsis (Arabidopsis thaliana) is described. Most PATs are expressed at ubiquitous levels and tissues throughout the development, while few genes are expressed especially during flower development preferentially in pollen and stamen. The proteins display large sequence and structural variations but exhibit a common protein topology that is preserved in PATs from various organisms. Arabidopsis PAT proteins display a complex targeting pattern and were detected at the endoplasmic reticulum, Golgi, endosomal compartments, and the vacuolar membrane. However, most proteins were targeted to the plasma membrane. This large concentration of plant PAT activity to the plasma membrane suggests that the plant cellular S-acylation machinery is functionally different compared with that of yeast (Saccharomyces cerevisiae) and mammalians.

Arp2/3 promotes junction formation and maintenance in theCaenorhabditis elegansintestine by regulating membrane association of apical proteins

It has been proposed that Arp2/3, which promotes nucleation of branched actin, is needed for epithelial junction initiation but is less important as junctions mature. We focus here on how Arp2/3 contributes to the Caenorhabditis elegans intestinal epithelium and find important roles for Arp2/3 in the maturation and maintenance of junctions in embryos and adults. Electron microscope studies show that embryos depleted of Arp2/3 form apical actin-rich microvilli and electron-dense apical junctions. However, whereas apical/basal polarity initiates, apical maturation is defective, including decreased apical F-actin enrichment, aberrant lumen morphology, and reduced accumulation of some apical junctional proteins, including DLG-1. Depletion of Arp2/3 in adult animals leads to similar intestinal defects. The DLG-1/AJM-1 apical junction proteins, and the ezrin-radixin-moesin homologue ERM-1, a protein that connects F-actin to membranes, are required along with Arp2/3 for apical F-actin enrichment in embryos, whereas cadherin junction proteins are not. Arp2/3 affects the subcellular distribution of DLG-1 and ERM-1. Loss of Arp2/3 shifts both ERM-1 and DLG-1 from pellet fractions to supernatant fractions, suggesting a role for Arp2/3 in the distribution of membrane-associated proteins. Thus, Arp2/3 is required as junctions mature to maintain apical proteins associated with the correct membranes.

Phosphorylation of the Par-1 polarity kinase by protein kinase D regulates 14-3-3 binding and membrane association.

The Par-1 protein kinases are conserved from yeast to humans, where they function as key polarity determinants. The mammalian Par-1 family is comprised of 4 members (Par-1a, -b, -c, and -d). Previously, we demonstrated that atypical protein kinase C (aPKC) phosphorylates the Par-1 kinases on a conserved threonine residue (T595) to regulate localization and kinase activity. Here, we demonstrate that Par-1b is also regulated by another arm of the PKC pathway, one that involves novel PKCs (nPKC) and protein kinase D. Treatment of cells with the PKC activator phorbol-12-myristate-13-acetate (PMA) potently stimulated phosphorylation of Par-1b on serine 400 (S400), a residue that is conserved in all 4 mammalian Par-1 kinases as well as the fly ortholog. We demonstrate that PMA stimulates nPKC to activate PKD, which in turn directly phosphorylates Par-1b on S400 to positively regulate 14-3-3 binding and to negatively regulate membrane association. Thus, 2 arms of the PKC pathway regulate interactions between Par-1b and 14-3-3 proteins: one involving aPKC and the other nPKC/PKD.

AAA ATPases regulate membrane association of yeast oxysterol binding proteins and sterol metabolism

The yeast genome encodes seven oxysterol binding protein homologs, Osh1p-Osh7p, which have been implicated in regulating intracellular lipid and vesicular transport. Here, we show that both Osh6p and Osh7p interact with Vps4p, a member of the AAA (ATPases associated with a variety of cellular activities) family. The coiled-coil domain of Osh7p was found to interact with Vps4p in a yeast two-hybrid screen and the interaction between Osh7p and Vps4p appears to be regulated by ergosterol. Deletion of VPS4 induced a dramatic increase in the membrane-associated pools of Osh6p and Osh7p and also caused a decrease in sterol esterification, which was suppressed by overexpression of OSH7. Lastly, overexpression of the coiled-coil domain of Osh7p (Osh7pCC) resulted in a multivesicular body sorting defect, suggesting a dominant negative role of Osh7pCC possibly through inhibiting Vps4p function. Our data suggest that a common mechanism may exist for AAA proteins to regulate the membrane association of yeast OSBP proteins and that these two protein families may function together to control subcellular lipid transport.

Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival

The abilities of mutated active RAS proteins to modulate cell survival following exposure to ionizing radiation and small molecule kinase inhibitors were examined. Homologous recombination in HCT116 cells to delete the single allele of K-RAS D13 resulted in a cell line that exhibited an approximately 75% reduction in basal extracellular signal-regulated kinase 1/2, AKT, and c-jun-NH2-kinase 1/2 activity. Transfection of cells lacking K-RAS D13 with H-RAS V12 restored extracellular signal-regulated kinase 1/2 and AKT activity to basal levels but did not restore c-jun-NH2-kinase 1/2 phosphorylation. In cells expressing H-RAS V12, radiation caused prolonged intense activation of AKT. Inhibition of H-RAS V12 function, blockade of phosphatidylinositol 3-kinase (PI3K) function using small interfering RNA/small-molecule inhibitors, or expression of dominant-negative AKT abolished radiation-induced AKT activation, and radiosensitized these cells. Inhibition of PI3K function did not significantly radiosensitize parental HCT116 cells. Inhibitors of the AKT PH domain including perifosine, SH-(5, 23-25) and ml-(14-16) reduced the plating efficiency of H-RAS V12 cells in a dose-dependent fashion. Inhibition of AKT function using perifosine enhanced radiosensitivity in H-RAS V12 cells, whereas the SH and ml series of AKT PH domain inhibitors failed to promote radiation toxicity. In HCT116 H-RAS V12 cells, PI3K, PDK-1, and AKT were membrane associated, whereas in parental cells expressing K-RAS D13, only PDK-1 was membrane bound. In H-RAS V12 cells, membrane associated PDK-1 was phosphorylated at Y373/376, which was abolished by the Src family kinase inhibitor PP2. Inhibition of PDK-1 function using the PH domain inhibitor OSU-03012 or using PP2 reduced the plating efficiency of H-RAS V12 cells and profoundly increased radiosensitivity. OSU-03012 and PP2 did not radiosensitize and had modest inhibitory effects on plating efficiency in parental cells. A small interfering RNA generated against PDK1 also radiosensitized HCT116 cells expressing H-RAS V12. Collectively, our data argue that molecular inhibition of AKT and PDK-1 signaling enhances the radiosensitivity of HCT116 cells expressing H-RAS V12 but not K-RAS D13. Small-molecule inhibitory agents that blocked stimulated and/or basal PDK-1 and AKT function profoundly reduced HCT116 cell survival but had variable effects at enhancing tumor cell radiosensitivity.

The Cytosolic Phospholipase A2 Catalytic Domain Modulates Association and Residence Time at Golgi Membranes

Cytosolic phospholipase A2 (cPLA2) catalyzes release of arachidonic acid from membranes following translocation to Golgi and endoplasmic reticulum. In response to an intracellular calcium concentration ([Ca2+]i) increase, the C2 domain binds Ca2+ and brings the catalytic domain into proximity with its phospholipid substrate. Because membrane residence is important in the regulation of cPLA2 activity, we explored the contributions of the C2 and catalytic domains in mediating membrane residence using an imaging approach in live cells with fluorescent protein chimeras of cPLA2. The isolated cPLA2 C2 domain associated with Golgi membranes rapidly in proportion to the [Ca2+]i, allowing for its use as a [Ca2+]i indicator. cPLA2 association with Golgi was slower than the isolated C2 domain in response to a [Ca2+]i increase. After [Ca2+]i decrease, cPLA2 remained associated with membrane in a Ca(2+)-independent fashion whereas C2 domain rapidly dissociated. Ca(2+)-independent membrane association was greatly reduced by mutation of Trp464, located at the membrane-exposed face of the catalytic domain, to Gly or Ala. Mutation of Trp464 to Phe supported Ca(2+)-independent association similar to wild type. These results demonstrate a role for the cPLA2 catalytic domain in regulating membrane association and membrane residence time.

Dominant-negative mutant of BIG2, an ARF–guanine nucleotide exchange factor, specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF–GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.

Overexpression of an ADP-ribosylation Factor-Guanine Nucleotide Exchange Factor, BIG2, Uncouples Brefeldin A-induced Adaptor Protein-1 Coat Dissociation and Membrane Tubulation

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.

Into the Membrane

BIOCHEMISTRY C2 domains are membrane-docking modules found in many signal transduction and vesicle-trafficking proteins. In many cases, calcium binding regulates membrane association. Murray and Honig used finite-difference Poisson-Boltzmann calculations to describe the electrostatic interactions of C2 domains of known structure with phospholipid membranes. Nonspecific electrostatic interactions were important for association with both negatively charged and neutral membranes. Calcium provided positive charge that attracted negatively charged membranes and reduced the desolvation penalty associated with membrane binding. Desolvation effects were particularly important in penetration of neutral membranes that in turn facilitate hydrophobic interactions. Calcium-independent C2 domains had similar electrostatic profiles. Quantitative estimates of the effect of experimental variables such as lipid composition, ionic strength, and mutations support a general role for these nonspecific electrostatic interactions. — VV Mol. Cell 9 , 145 (2002).

The Vps4p AAA ATPase regulates membrane association of a Vps protein complex required for normal endosome function.

Vps4p is an AAA-type ATPase required for efficient transport of biosynthetic and endocytic cargo from an endosome to the lysosome-like vacuole of Saccharomyces cerevisiae. Vps4p mutants that do not bind ATP or are defective in ATP hydrolysis were characterized both in vivo and in vitro. The nucleotide-free or ADP-bound form of Vps4p existed as a dimer, whereas in the ATP-locked state, Vps4p dimers assembled into a decameric complex. This suggests that ATP hydrolysis drives a cycle of association and dissociation of Vps4p dimers/decamers. Nucleotide binding also regulated the association of Vps4p with an endosomal compartment in vivo. This membrane association required the N-terminal coiled-coil motif of Vps4p, but deletion of the coiled-coil domain did not affect ATPase activity or oligomeric assembly of the protein. Membrane association of two previously uncharacterized class E Vps proteins, Vps24p and Vps32p/Snf7p, was also affected by mutations in VPS4. Upon inactivation of a temperature-conditional vps4 mutant, Vps24p and Vps32p/Snf7p rapidly accumulated in a large membrane-bound complex. Immunofluorescence indicated that both proteins function with Vps4p at a common endosomal compartment. Together, the data suggest that the Vps4 ATPase catalyzes the release (uncoating) of an endosomal membrane-associated class E protein complex(es) required for normal morphology and sorting activity of the endosome.

SHIP Modulates Immune Receptor Responses by Regulating Membrane Association of Btk

Membrane recruitment of SHIP is responsible for the inhibitory signal generated by FcγRIIB coligation to the BCR. By reducing the level of PIP3, SHIP regulates the association of the tyrosine kinase Btk with the membrane through PH domain–phosphoinositol lipid interactions. Inhibition of BCR signaling by either FcγRIIB coligation, membrane expression of SHIP, or inhibition of PI3K, conditions which result in decreased levels of PIP3, is suppressed by the expression of Btk as a membrane-associated chimera. Conversely, increasing PIP3 levels by deletion of SHIP results in increased Btk association with the membrane and hyperresponsive BCR signaling. These results suggest a central role for PIP3 in regulating the B cell stimulatory state by modulating Btk localization and thereby calcium fluxes.