Biomedical subjects

Maurine E Linder

Publications and source records attributed to Maurine E Linder.

18 recordsLinked to original sources

PubMed2007

Palmitoylation: policing protein stability and traffic.

Palmitate modifies both peripheral and integral membrane proteins and its addition can be permanent or transient, which makes it unique among the lipid modifications of proteins. The presence of palmitate on a protein affects how the protein interacts with lipids and proteins in a membrane compartment, and the reversibility of palmitoylation allows different modes of trafficking between membrane compartments. Here, we review recent studies that have provided insights into the mechanisms that mediate the functional consequences of this versatile modification.

Acylation↗

PubMed2006

R7BP augments the function of RGS7*Gbeta5 complexes by a plasma membrane-targeting mechanism.

The RGS7 (R7) family of G protein regulators, Gbeta5, and R7BP form heterotrimeric complexes that potently regulate the kinetics of G protein-coupled receptor signaling. Reversible palmitoylation of R7BP regulates plasma membrane/nuclear shuttling of R7*Gbeta5*R7BP heterotrimers. Here we have investigated mechanisms whereby R7BP controls the function of the R7 family. We show that unpalmitoylated R7BP undergoes nuclear/cytoplasmic shuttling and that a C-terminal polybasic motif proximal to the palmitoylation acceptor sites of R7BP mediates nuclear localization, palmitoylation, and plasma membrane targeting. These results suggest a novel mechanism whereby palmitoyltransferases and nuclear import receptors both utilize the C-terminal domain of R7BP to determine the trafficking fate of R7*Gbeta5*R7BP heterotrimers. Analogous mechanisms may regulate other signaling proteins whose distribution between the plasma membrane and nucleus is controlled by palmitoylation. Lastly, we show that cytoplasmic RGS7*Gbeta5*R7BP heterotrimers and RGS7*Gbeta5 heterodimers are equivalently inefficient regulators of G protein-coupled receptor signaling relative to plasma membrane-bound heterotrimers bearing palmitoylated R7BP. Therefore, R7BP augments the function of the complex by a palmitoylation-regulated plasma membrane-targeting mechanism.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Protein palmitoylation by a family of DHHC protein S-acyltransferases.

Protein palmitoylation refers to the posttranslational addition of a 16 carbon fatty acid to the side chain of cysteine, forming a thioester linkage. This acyl modification is readily reversible, providing a potential regulatory mechanism to mediate protein-membrane interactions and subcellular trafficking of proteins. The mechanism that underlies the transfer of palmitate or other long-chain fatty acids to protein was uncovered through genetic screens in yeast. Two related S-palmitoyltransferases were discovered. Erf2 palmitoylates yeast Ras proteins, whereas Akr1 modifies the yeast casein kinase, Yck2. Erf2 and Akr1 share a common sequence referred to as a DHHC (aspartate-histidine-histidine-cysteine) domain. Numerous genes encoding DHHC domain proteins are found in all eukaryotic genome databases. Mounting evidence is consistent with this signature motif playing a direct role in protein acyltransferase (PAT) reactions, although many questions remain. This review presents the genetic and biochemical evidence for the PAT activity of DHHC proteins and discusses the mechanism of protein-mediated palmitoylation.

Acetyltransferases↗

PubMed2006

Biochemical characterization of RGS14: RGS14 activity towards G-protein alpha subunits is independent of its binding to Rap2A.

RGS (regulators of G-protein signalling) modulate signalling by acting as GAPs (GTPase-activating proteins) for alpha subunits of heterotrimeric G-proteins. RGS14 accelerates GTP hydrolysis by G(ialpha) family members through its RGS domain and suppresses guanine nucleotide dissociation from G(ialpha1) and G(ialpha3) subunits through its C-terminal GoLoco domain. Additionally, RGS14 binds the activated forms of the small GTPases Rap1 and Rap2 by virtue of tandem RBDs (Raf-like Ras/Rap binding domains). RGS14 was identified in a screen for Rap2 effectors [Traver, Splingard, Gaudriault and De Gunzburg (2004) Biochem. J. 379, 627-632]. In the present study, we tested whether Rap binding regulates RGS14's biochemical activities. We found that RGS14 activity towards heterotrimeric G-proteins, as either a GAP or a GDI (guanine nucleotide dissociation inhibitor), was unaffected by Rap binding. Extending our biochemical characterization of RGS14, we also examined whether RGS14 can suppress guanine nucleotide exchange on G(ialpha1) in the context of the heterotrimer. We found that a heterotrimer composed of N-myristoylated G(ialpha1) and prenylated G(betagamma) is resistant to the GDI activity of the GoLoco domain of RGS14. This is consistent with models of GoLoco domain action on free G(alpha) and suggests that RGS14 alone cannot induce subunit dissociation to promote receptor-independent activation of G(betagamma)-mediated signalling pathways.

Animals↗

PubMed2006

Purification and characterization of recombinant protein acyltransferases.

Palmitoylation enhances membrane association and plays a role in the subcellular trafficking and signaling function of proteins. Unlike other forms of protein lipidation, such as prenylation and myristoylation, palmitoylation is reversible and can therefore play a regulatory role. Enzyme activities have recently been described in mammals and yeast that carry out the palmitoylation of protein substrates. Protein acyltransferases (PATs) transfer a palmitoyl moiety derived from palmitoyl-CoA to a free thiol of a substrate protein to create a labile thioester linkage. Biochemical characterization and kinetic analysis of this new family of enzymes requires methods to purify PATs and their substrates, as well as methods to assay PAT activity. We describe a series of methods using yeast and bacterial expression systems to study protein acyltransferases.

Acyltransferases↗

PubMed2005

The vacuolar DHHC-CRD protein Pfa3p is a protein acyltransferase for Vac8p.

Palmitoylation of the vacuolar membrane protein Vac8p is essential for vacuole fusion in yeast (Veit, M., R. Laage, L. Dietrich, L. Wang, and C. Ungermann. 2001. EMBO J. 20:3145-3155; Wang, Y.X., E.J. Kauffman, J.E. Duex, and L.S. Weisman. 2001. J. Biol. Chem. 276:35133-35140). Proteins that contain an Asp-His-His-Cys (DHHC)-cysteine rich domain (CRD) are emerging as a family of protein acyltransferases, and are therefore candidates for mediators of Vac8p palmitoylation. Here we demonstrate that the DHHC-CRD proteins Pfa3p (protein fatty acyltransferase 3, encoded by YNL326c) and Swf1p are important for vacuole fusion. Cells lacking Pfa3p had fragmented vacuoles when stressed, and cells lacking both Pfa3p and Swf1p had fragmented vacuoles under normal growth conditions. Pfa3p promoted Vac8p membrane association and palmitoylation in vivo and partially purified Pfa3p palmitoylated Vac8p in vitro, establishing Vac8p as a substrate for palmitoylation by Pfa3p. Vac8p is the first N-myristoylated, palmitoylated protein identified as a substrate for a DHHC-CRD protein.

Acyltransferases↗

PubMed2005

DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras.

Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

Acyltransferases↗

PubMed2005

Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family.

The RGS7 (R7) family of RGS proteins bound to the divergent Gbeta subunit Gbeta5 is a crucial regulator of G protein-coupled receptor (GPCR) signaling in the visual and nervous systems. Here, we identify R7BP, a novel neuronally expressed protein that binds R7-Gbeta5 complexes and shuttles them between the plasma membrane and nucleus. Regional expression of R7BP, Gbeta5, and R7 isoforms in brain is highly coincident. R7BP is palmitoylated near its COOH terminus, which targets the protein to the plasma membrane. Depalmitoylation of R7BP translocates R7BP-R7-Gbeta5 complexes from the plasma membrane to the nucleus. Compared with nonpalmitoylated R7BP, palmitoylated R7BP greatly augments the ability of RGS7 to attenuate GPCR-mediated G protein-regulated inward rectifying potassium channel activation. Thus, by controlling plasma membrane nuclear-shuttling of R7BP-R7-Gbeta5 complexes, reversible palmitoylation of R7BP provides a novel mechanism that regulates GPCR signaling and potentially transduces signals directly from the plasma membrane to the nucleus.

Active Transport, Cell Nucleus↗

PubMed2004

The RGS14 GoLoco domain discriminates among Galphai isoforms.

Regulators of G protein signaling (RGS) modulate G protein activity by functioning as GTPase-activating proteins (GAPs) for alpha-subunits of heterotrimeric G proteins. RGS14 regulates G protein nucleotide exchange and hydrolysis by acting as a GAP through its RGS domain and as a guanine nucleotide dissociation inhibitor (GDI) through its GoLoco motif. RGS14 exerts GDI activity on Galphai1, but not Galphao. Selective interactions are mediated by contacts between the alphaA and alphaB helices of the Galphai1 helical domain and the GoLoco C terminus (Kimple, R. J., Kimple, M. E., Betts, L., Sondek, J., and Siderovski, D. P. (2002) Nature 416, 878-881). Three isoforms of Galphai exist in mammalian cells. In this study, we tested whether all three isoforms were subject to RGS14 GDI activity. We found that RGS14 inhibits guanine nucleotide exchange on Galphai1 and Galphai3 could, but not Galphai2. Galphai2 be rendered sensitive to RGS14 GDI activity by replacement of residues within the alpha-helical domain. In addition to the contact residues in the alphaA and alphaB helices previously identified, we found that the alphaA/alphaB and alphaB/alphaC loops are important determinants of Galphai selectivity. The striking selectivity observed for RGS14 GDI activity in vitro points to Galphai1 and Galphai3 as the likely targets of RGS14-GoLoco regulation in vivo.

Amino Acid Motifs↗

PubMed2004

Reciprocal signaling between the transcriptional co-factor Eya2 and specific members of the Galphai family.

As part of a program to elucidate signaling processes controlled by the heterotrimeric G protein Galphaz, a human fetal brain cDNA library was screened for proteins that specifically interact with the activated form of Galphaz. One of the most-encountered molecules in this screen was Eya2, a member of the Eyes absent family of proteins. Mammalian Eya proteins are predominantly cytosolic proteins that are known to interact with members of the Sine oculis (Six) family of homeodomain transcription factors. This interaction facilitates the translocation of Eya into the nucleus, where the Eya/Six complex regulates transcription during critical stages of embryonic development. In vitro binding studies confirmed that Galphaz interacts with Eya2 in an activation-dependent fashion; furthermore, most other members of the Galphai family including Galphai1, Galphai2, and Galphai3 were found to interact with Eya2. It is interesting that one of the most abundant Galphai proteins, Galphao, did not interact with Eya2. Coexpression of the activated forms of Galphai1, Galphai2, and Galphai3, but not Galphao, with Eya2 recruited Eya2 to the plasma membrane, prevented Eya2 translocation into the nucleus, and abrogated Eya2/Six4-mediated transcription. In addition, Eya2 impinged on G protein-mediated signaling, as evidenced by its ability to relieve Galphai2-mediated inhibition of adenylyl cyclase. These results demonstrate that the interaction between the Galphai proteins and Eya2 may impact on seemingly disparate regulatory events involving both classes of proteins.

Adenylyl Cyclase Inhibitors↗

PubMed2004

Model organisms lead the way to protein palmitoyltransferases.

The acylation of proteins with palmitate and related fatty acids has been known for over 30 years, but the molecular machinery that carries out palmitoylation has only recently emerged from studies in the model organisms Saccharomyces cerevisiae and Drosophila. Two classes of protein acyltransferases (PATs) have been proposed. In yeast, members of a family of integral membrane proteins harboring a cysteine-rich domain (CRD) containing a conserved DHHC (Asp-His-His-Cys) motif are PATs for cytoplasmic signaling molecules. The DHHC-CRD protein Erf2p, together with an associated subunit Erf4p, palmitoylates yeast Ras proteins, and Akr1p catalyzes the palmitoylation of the yeast casein kinase Yck2p. The existence of a second class of PATs that modify secreted signaling proteins has been suggested from work in Drosophila. Rasp is required in vivo for the production of functional Hedgehog and shares sequence identity with membrane-bound O-acyltransferases, which suggests that it catalyzes the palmitoylation of Hedgehog. With the identification of PATs in model genetic organisms, the field is now poised to uncover their mammalian counterparts and to understand the enzymology of protein palmitoylation.

Acetyltransferases↗

PubMed2004

Palmitoylation of intracellular signaling proteins: regulation and function.

Protein S-palmitoylation is the thioester linkage of long-chain fatty acids to cysteine residues in proteins. Addition of palmitate to proteins facilitates their membrane interactions and trafficking, and it modulates protein-protein interactions and enzyme activity. The reversibility of palmitoylation makes it an attractive mechanism for regulating protein activity, and this feature has generated intensive investigation of this modification. The regulation of palmitoylation occurs through the actions of protein acyltransferases and protein acylthioesterases. Identification of the protein acyltransferases Erf2/Erf4 and Akr1 in yeast has provided new insight into the palmitoylation reaction. These molecules work in concert with thioesterases, such as acyl-protein thioesterase 1, to regulate the palmitoylation status of numerous signaling molecules, ultimately influencing their function. This review discusses the function and regulation of protein palmitoylation, focusing on intracellular proteins that participate in cell signaling or protein trafficking.

Acylation↗

PubMed2004

Analysis of RGS protein palmitoylation.

Palmitoylation refers to the covalent attachment of a 16-carbon fatty acid to cysteine residues of proteins. This modification occurs on many intracellular signaling proteins including regulators of G protein signaling proteins (RGS). Palmitoylation mediates the interaction of proteins with membranes and other proteins and can control the biological activity of a protein. Palmitate attachment occurs through a labile thioester bond and is readily reversible in cells, thus providing a particularly important means for protein regulation. This chapter presents protocols for investigating RGS protein palmitoylation in mammalian cells. The RGS protein of interest is heterologously expressed in HEK293 cells, and cells are metabolically labeled with [3H]palmitate. The RGS protein is isolated from fractionated cells by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography to determine if [3H] has been incorporated. To confirm that the radiolabeled fatty acid is linked to the protein through a thioester bond, labeled proteins are treated with neutral hydroxylamine. Oxyester-linked palmitate, which is occasionally found on serine and threonine residues, is insensitive to this treatment, whereas thioesters are sensitive. To verify that incorporated radiolabel is palmitate, the protein is treated with base, which also cleaves thioester bonds. The resulting lipids are extracted from the sample, then analyzed by chromatography.

Acylation↗

PubMed2004

Purification of recombinant G protein alpha subunits from Escherichia coli.

The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.

Chromatography, Affinity↗

PubMed2003

New insights into the mechanisms of protein palmitoylation.

Since its discovery more than 30 years ago, protein palmitoylation has been shown to have a role in protein-membrane interactions, protein trafficking, and enzyme activity. Until recently, however, the molecular machinery that carries out reversible palmitoylation of proteins has been elusive. In fact, both enzymatic and nonenzymatic S-acylation reaction mechanisms have been proposed. Recent reports of protein palmitoyltransferases in Saccharomyces cerevisiae and Drosophila provide the first glimpse of enzymes that carry out protein palmitoylation. Equally important is the mechanism of depalmitoylation. Two major classes of protein palmitoylthioesterases have been described. One family is lysosomal and is involved in protein degradation. The second is cytosolic and removes palmitoyl moieties preferentially from proteins associated with membranes. This review discusses recent advances in the understanding of mechanisms of addition of palmitate to proteins and removal of palmitate from proteins.

Acetyltransferases↗

PubMed2002

Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.

Most Ras proteins are posttranslationally modified by a palmitoyl lipid moiety through a thioester linkage. However, the mechanism by which this occurs is not known. Here, evidence is presented that the Ras2 protein of Saccharomyces cerevisiae is palmitoylated by a Ras protein acyltransferase (Ras PAT) encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the membrane of the endoplasmic reticulum and contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Erf2p co-purifies with Erf4p (26 kDa) when it is expressed in yeast or in Escherichia coli. The Erf2p/Erf4p complex is required for Ras PAT activity, and mutations within conserved residues (Cys(189), His(201), and Cys(203)) of the Erf2p DHHC-CRD domain abolish Ras PAT activity. Furthermore, a palmitoyl-Erf2p intermediate is detected suggesting that Erf2p is directly involved in palmitate transfer. ERF2 and ERF4 are the first genes identified that encode a palmitoyltransferase for a Ras GTPase.

Genes, Fungal↗

PubMed2002

Distinct sites on G protein beta gamma subunits regulate different effector functions.

G proteins interact with effectors at multiple sites and regulate their activity. The functional significance of multiple contact points is not well understood. We previously identified three residues on distinct surfaces of Gbetagamma that are crucial for G protein-coupled inward rectifier K(+) (GIRK) channel activation. Here we show that mutations at these sites, S67K, S98T, and T128F, abolished or reduced direct GIRK current activation in inside-out patches, but, surprisingly, all mutants synergized with sodium in activating K(+) currents. Each of the three Gbeta(1) mutants bound the channel indicating that the defects reflected mainly functional impairments. We tested these mutants for functional interactions with effectors other than K(+) channels. With N-type calcium channels, Gbetagamma wild type and mutants all inhibited basal currents. A depolarizing pre-pulse relieved Gbetagamma inhibition of Ca(2+) currents by the wild type and the S98T and T128F mutants but not the S67K mutant. Both wild type and mutant Gbetagamma subunits activated phospholipase C beta(2) with similar potencies; however, the S67K mutant showed reduced maximal activity. These data establish a pattern where mutations can alter the Gbetagamma regulation of a specific effector function without affecting other Gbetagamma-mediated functions. Moreover, Ser-67 showed this pattern in all three effectors tested, suggesting that this residue participates in a common functional domain on Gbeta(1) that regulates several effectors. These data show that distinct domains within Gbetagamma subserve specific functional roles.

Animals↗

PubMed2002

SNAP-25 traffics to the plasma membrane by a syntaxin-independent mechanism.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are essential for vesicle docking and fusion. SNAP-25, syntaxin 1A, and synaptobrevin/vesicle-associated membrane protein (VAMP) are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. It has been proposed that interactions of SNAP-25 with syntaxin 1A are required for initial membrane attachment of SNAP-25 (Vogel, K., Cabaniols, J.-P., and Roche, P. (2000) J. Biol. Chem. 275, 2959-2965). However, we have shown previously that residues 85-120 of the SNAP-25 interhelical domain, which do not interact with syntaxin, are necessary and sufficient for palmitoylation and plasma membrane localization of a green fluorescent protein reporter molecule (Gonzalo, S., Greentree, W. K., and Linder, M. E. (1999) J. Biol. Chem. 274, 21313-21318). To clarify the role of syntaxin in membrane targeting of SNAP-25, we studied a SNAP-25 point mutant (G43D) that does not interact with syntaxin. SNAP-25 G43D/green fluorescent protein was palmitoylated and localized at the plasma membrane. Newly synthesized SNAP-25 G43D had the same kinetics of membrane association as the wild-type protein. Furthermore, expression of a cytosolic mutant syntaxin 1A did not interfere with SNAP-25 membrane interactions or palmitoylation in the neuronal cell line NG108-15. Exogenously expressed SNAP-25 targets efficiently to the plasma membrane in cells of neuronal origin but only partially in HeLa cells, a neurosecretion-incompetent line. This phenotype was not rescued when syntaxin 1A was co-expressed with SNAP-25. Our data support a syntaxin-independent mechanism of membrane targeting for SNAP-25.

Biological Transport↗