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1.
Dr Piers A. Hemsley 《Molecular membrane biology》2013,30(1-2):114-125
Membrane resident proteins are a common feature of biology yet many of these proteins are not integral to the membrane. These peripheral membrane proteins are often bound to the membrane by the addition of fatty acyl chains to the protein. This modification, known as S-acylation or palmitoylation, promotes very strong membrane association but is also reversible allowing for a high degree of control over membrane association. Many S-acylated proteins are resident in sterol, sphingolipid and saturated-lipid enriched microdomains indicating an important role for S-acylation in protein partitioning within membranes. This review summarises the current knowledge of S-acylation in plants. S-acylated proteins play a wide variety of roles in plants and affect Ca2+ signalling, K+ movement, stress signalling, small and heterotrimeric G-protein membrane association and partitioning, tubulin function as well as pathogenesis. Although the study of S-acylation is in its infancy in plants this review illustrates that S-acylation is extremely important for plant function and that there are many unexplored aspects of S-acylation in plants. A full summary of the techniques and methods available to study S-acylation in plants is also presented. 相似文献
2.
Gorleku OA Barns AM Prescott GR Greaves J Chamberlain LH 《The Journal of biological chemistry》2011,286(45):39573-39584
Intracellular palmitoylation dynamics are regulated by a family of 24 DHHC (aspartate-histidine-histidine-cysteine) palmitoyltransferases, which are localized in a compartment-specific manner. The majority of DHHC proteins localize to endoplasmic reticulum (ER) and Golgi membranes, and a small number target to post-Golgi membranes. To date, there are no reports of the fine mapping of sorting signals in mammalian DHHC proteins; thus, it is unclear how spatial distribution of the DHHC family is achieved. Here, we have identified and characterized lysine-based sorting signals that determine the restricted localization of DHHC4 and DHHC6 to ER membranes. The ER targeting signal in DHHC6 conforms to a KKXX motif, whereas the signal in DHHC4 is a distinct KXX motif. The identified dilysine signals are sufficient to specify ER localization as adding the C-terminal pentapeptide sequences from DHHC4 or DHHC6, which contain these KXX and KKXX motifs, to the C terminus of DHHC3, redistributes this palmitoyltransferase from Golgi to ER membranes. Recent work proposed that palmitoylation of newly synthesized peripheral membrane proteins occurs predominantly at the Golgi. Indeed, previous analyses of the peripheral membrane proteins, SNAP25 and cysteine string protein, are fully consistent with their initial palmitoylation being mediated by Golgi-localized DHHC proteins. Interestingly, ER-localized DHHC3 is able to palmitoylate SNAP25 and cysteine string protein to a similar level as wild-type Golgi-localized DHHC3 in co-expression studies. These results suggest that targeting of intrinsically active DHHC proteins to defined membrane compartments is an important factor contributing to spatially restricted patterns of substrate palmitoylation. 相似文献
3.
Intracellular palmitoylation dynamics are regulated by a large family of DHHC (Asp-His-His-Cys) palmitoyl transferases. The majority of DHHC proteins associate with endoplasmic reticulum (ER) or Golgi membranes, but an interesting exception is DHHC2, which localizes to dendritic vesicles of unknown origin in neurons, where it regulates dynamic palmitoylation of PSD95. Dendritic targeting of newly synthesized PSD95 is likely preceded by palmitoylation on Golgi membranes by DHHC3 and/or DHHC15. The precise intracellular distribution of DHHC2 is presently unclear, and there is very little known in general about how DHHC proteins achieve their respective localizations. In this study, membrane targeting of DHHC2 in live and fixed neuroendocrine cells was investigated and mutational analysis employed to define regions of DHHC2 that regulate targeting. We report that DHHC2 associates with the plasma membrane, Rab11-positive recycling endosomes, and vesicular structures. Plasma membrane integration of DHHC2 was confirmed by labeling of an extrafacial HA epitope in nonpermeabilized cells. Antibody-uptake experiments suggested that DHHC2 traffics between the plasma membrane and intracellular membranes. This dynamic localization was confirmed using fluorescence recovery after photo-bleaching analysis, which revealed constitutive refilling of the recycling endosome (RE) pool of DHHC2. The cytoplasmic C-terminus of DHHC2 regulates membrane targeting and a mutant lacking this domain was associated with the ER. Although DHHC2 is closely related to DHHC15, these proteins populate distinct membrane compartments. Construction of chimeric DHHC2/DHHC15 proteins revealed that this difference in localization is a consequence of divergent sequences within their C-terminal tails. This study is the first to highlight dynamic cycling of a mammalian DHHC protein between clearly defined membrane compartments, and to identify domains that specify membrane targeting of this protein family. 相似文献
4.
Dual lipid modification of Arabidopsis Ggamma-subunits is required for efficient plasma membrane targeting
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Posttranslational lipid modifications are important for proper localization of many proteins in eukaryotic cells. However, the functional interrelationships between lipid modification processes in plants remain unclear. Here we demonstrate that the two heterotrimeric G-protein gamma-subunits from Arabidopsis (Arabidopsis thaliana), AGG1 and AGG2, are prenylated, and AGG2 is S-acylated. In wild type, enhanced yellow fluorescent protein-fused AGG1 and AGG2 are associated with plasma membranes, with AGG1 associated with internal membranes as well. Both can be prenylated by either protein geranylgeranyltransferase I (PGGT-I) or protein farnesyltransferase (PFT). Their membrane localization is intact in mutants lacking PFT activity and largely intact in mutants lacking PGGT-I activity but is disrupted in mutants lacking both PFT and PGGT-I activity. Unlike in mammals, Arabidopsis Ggammas do not rely on functional Galpha for membrane targeting. Mutation of the sixth to last cysteine, the putative S-acylation acceptor site, causes a dramatic change in AGG2 but not AGG1 localization pattern, suggesting S-acylation serves as an important additional signal for AGG2 to be targeted to the plasma membrane. Domain-swapping experiments suggest that a short charged sequence at the AGG2 C terminus contributes to AGG2's efficient membrane targeting compared to AGG1. Our data show the large degree to which PFT and PGGT-I can compensate for each other in plants and suggest that differential lipid modification plays an important regulatory role in plant protein localization. 相似文献
5.
Kanaani J el-Husseini Ael-D Aguilera-Moreno A Diacovo JM Bredt DS Baekkeskov S 《The Journal of cell biology》2002,158(7):1229-1238
The signals involved in axonal trafficking and presynaptic clustering are poorly defined. Here we show that targeting of the gamma-aminobutyric acid-synthesizing enzyme glutamate decarboxylase 65 (GAD65) to presynaptic clusters is mediated by its palmitoylated 60-aa NH(2)-terminal domain and that this region can target other soluble proteins and their associated partners to presynaptic termini. A Golgi localization signal in aa 1-23 followed by a membrane anchoring signal upstream of the palmitoylation motif are required for this process and mediate targeting of GAD65 to the cytosolic leaflet of Golgi membranes, an obligatory first step in axonal sorting. Palmitoylation of a third trafficking signal downstream of the membrane anchoring signal is not required for Golgi targeting. However, palmitoylation of cysteines 30 and 45 is critical for post-Golgi trafficking of GAD65 to presynaptic sites and for its relative dendritic exclusion. Reduction of cellular cholesterol levels resulted in the inhibition of presynaptic clustering of palmitoylated GAD65, suggesting that the selective targeting of the protein to presynaptic termini is dependent on sorting to cholesterol-rich membrane microdomains. The palmitoylated NH(2)-terminal region of GAD65 is the first identified protein region that can target other proteins to presynaptic clusters. 相似文献
6.
Connie B. Nichols Kyla S. Ost Dayton P. Grogan Kaila Pianalto Shirin Hasan J. Andrew Alspaugh 《Eukaryotic cell》2015,14(7):626-635
The localization and specialized function of Ras-like proteins are largely determined by posttranslational processing events. In a highly regulated process, palmitoyl groups may be added to C-terminal cysteine residues, targeting these proteins to specific membranes. In the human fungal pathogen Cryptococcus neoformans, Ras1 protein palmitoylation is essential for growth at high temperature but is dispensable for sexual differentiation. Ras1 palmitoylation is also required for localization of this protein on the plasma membrane. Together, these results support a model in which specific Ras functions are mediated from different subcellular locations. We therefore hypothesize that proteins that activate Ras1 or mediate Ras1 localization to the plasma membrane will be important for C. neoformans pathogenesis. To further characterize the Ras1 signaling cascade mediating high-temperature growth, we have identified a family of protein S-acyltransferases (PATs), enzymes that mediate palmitoylation, in the C. neoformans genome database. Deletion strains for each candidate gene were generated by homogenous recombination, and each mutant strain was assessed for Ras1-mediated phenotypes, including high-temperature growth, morphogenesis, and sexual development. We found that full Ras1 palmitoylation and function required one particular PAT, Pfa4, and deletion of the PFA4 gene in C. neoformans resulted in altered Ras1 localization to membranes, impaired growth at 37°C, and reduced virulence. 相似文献
7.
The tandem repeat domain in the Listeria monocytogenes ActA protein controls the rate of actin-based motility, the percentage of moving bacteria, and the localization of vasodilator-stimulated phosphoprotein and profilin 总被引:7,自引:0,他引:7
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《The Journal of cell biology》1996,135(3):647-660
A variety of cysteine-containing, lipid-modified peptides are found to be S-acylated by cultured mammalian cells. The acylation reaction is highly specific for cysteinyl over serinyl residues and for lipid- modified peptides over hydrophilic peptides. The S-acylation process appears by various criteria to be enzymatic and resembles the S- acylation of plasma membrane-associated proteins in various characteristics, including inhibition by tunicamycin. The substrate range of the S-acylation reaction encompasses, but is not limited to, lipopeptides incorporating the motifs myristoylGC- and -CXC(farnesyl)- OCH3, which are reversibly S-acylated in various intracellular proteins. Mass-spectrometric analysis indicates that palmitoyl residues constitute the predominant but not the only type of S-acyl group coupled to a lipopeptide carrying the myristoylGC- motif, with smaller amounts of S-stearoyl and S-oleoyl substituents also detectable. Fluorescence microscopy using NBD-labeled cysteinyl lipopeptides reveals that the products of lipopeptide S-acylation, which cannot diffuse between membranes, are in almost all cases localized preferentially to the plasma membrane. This preferential localization is found even at reduced temperatures where vesicular transport from the Golgi complex to the plasma membrane is suppressed, strongly suggesting that the plasma membrane itself is the preferred site of S- acylation of these species. Uniquely among the lipopeptides studied, species incorporating an unphysiological N-myristoylcysteinyl- motif also show substantial formation of S-acylated products in a second, intracellular compartment identified as the Golgi complex by its labeling with a fluorescent ceramide. Our results suggest that distinct S-acyltransferases exist in the Golgi complex and plasma membrane compartments and that S-acylation of motifs such as myristoylGC- occurs specifically at the plasma membrane, affording efficient targeting of cellular proteins bearing such motifs to this membrane compartment. 相似文献
8.
Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association. 总被引:6,自引:1,他引:5
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Members of the Src family of protein tyrosine kinases are localized to subspecialized regions of the plasma membrane. Herein we show that the N-terminal SH4 region of the Src family member p59fyn (Fyn) is both necessary and sufficient for targeting of Fyn and heterologous proteins to the plasma membrane and detergent-insoluble subdomains. Attachment of the first 16 amino acids of Fyn to a normally cytosolic protein, beta-galactosidase, resulted in distinct plasma membrane localization of the chimeric protein. Mutation of the palmitoylation site (cysteine-3) within Fyn16-beta-galactosidase or wild-type Fyn abrogated plasma membrane localization, resulting in redistribution of the mutant proteins into intracellular membranes. Substitution of the SH4 motif within Fyn with heterologous sequences from other palmitoylated proteins (G alpha o and GAP43) revealed that the presence of palmitate is sufficient to direct plasma membrane localization independent of surrounding amino acid sequences and myristate. Palmitoylated Fyn chimeras were also enriched in the Triton X-100-resistant matrix, whereas nonpalmitoylated forms of these proteins were detected in the detergent-soluble fraction. The palmitate moiety on Fyn exhibited a half-life of 1.5-2 h. In contrast, the half-life of the polypeptide backbone was 8 h, indicating that palmitoylation is a reversible modification. These studies establish that the palmitoylated SH4 sequence of Fyn can be used to specifically target proteins to the plasma membrane in a reversible manner. 相似文献
9.
Kenworthy AK 《Methods (San Diego, Calif.)》2006,40(2):198-205
A well known function of palmitoylation is to promote protein binding to cell membranes. Until recently, it was unclear what additional roles, if any, palmitoylation has in controlling protein localization in cells. Recent studies of palmitoylated forms of the small GTPase Ras have now revealed that palmitoylation plays multiple roles in the regulation of protein trafficking, including targeting proteins into the secretory pathway and recycling proteins between the plasma membrane and Golgi complex. We here describe how quantitative fluorescence microscopy and photobleaching approaches can be used to study the intracellular targeting and trafficking of GFP-tagged palmitoylated proteins in living cells. We discuss (1) general considerations for fluorescence recovery after photobleaching (FRAP) measurements of GFP-tagged proteins; (2) FRAP-based assays to test the strength of binding of palmitoylated proteins to cell membranes; (3) methods to establish the kinetics and mechanisms of recycling of palmitoylated proteins between the Golgi complex and the plasma membrane; (4) the use of the palmitoylation inhibitor 2-bromo-palmitate as a tool to study the dynamic regulation of protein targeting and trafficking by palmitate turnover. 相似文献
10.
Dual fatty acyl modification determines the localization and plasma membrane targeting of CBL/CIPK Ca2+ signaling complexes in Arabidopsis 总被引:2,自引:0,他引:2
Arabidopsis thaliana calcineurin B-like proteins (CBLs) interact specifically with a group of CBL-interacting protein kinases (CIPKs). CBL/CIPK complexes phosphorylate target proteins at the plasma membrane. Here, we report that dual lipid modification is required for CBL1 function and for localization of this calcium sensor at the plasma membrane. First, myristoylation targets CBL1 to the endoplasmic reticulum. Second, S-acylation is crucial for endoplasmic reticulum-to-plasma membrane trafficking via a novel cellular targeting pathway that is insensitive to brefeldin A. We found that a 12-amino acid peptide of CBL1 is sufficient to mediate dual lipid modification and to confer plasma membrane targeting. Moreover, the lipid modification status of the calcium sensor moiety determines the cellular localization of preassembled CBL/CIPK complexes. Our findings demonstrate the importance of S-acylation for regulating the spatial accuracy of Ca2+-decoding proteins and suggest a novel mechanism that enables the functional specificity of calcium sensor/kinase complexes. 相似文献
11.
New insights into the mechanisms of protein palmitoylation 总被引:11,自引:0,他引:11
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. 相似文献
12.
Evanko DS Thiyagarajan MM Wedegaertner PB 《The Journal of biological chemistry》2000,275(2):1327-1336
Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits. 相似文献
13.
Palmitoylation and membrane interactions of the neuroprotective chaperone cysteine-string protein 总被引:1,自引:0,他引:1
Greaves J Salaun C Fukata Y Fukata M Chamberlain LH 《The Journal of biological chemistry》2008,283(36):25014-25026
Cysteine-string protein (CSP) is an extensively palmitoylated DnaJ-family chaperone, which exerts an important neuroprotective function. Palmitoylation is required for the intracellular sorting and function of CSP, and thus it is important to understand how this essential modification of CSP is regulated. Recent work identified 23 putative palmitoyl transferases containing a conserved DHHC domain in mammalian cells, and here we show that palmitoylation of CSP is enhanced specifically by co-expression of the Golgi-localized palmitoyl transferases DHHC3, DHHC7, DHHC15, or DHHC17. Indeed, these DHHC proteins promote stable membrane attachment of CSP, which is otherwise cytosolic. An inverse correlation was identified between membrane affinity of unpalmitoylated CSP mutants and subsequent palmitoylation: mutants with an increased membrane affinity localize to the endoplasmic reticulum (ER) and are physically separated from the Golgi-localized DHHC proteins. Palmitoylation of an ER-localized mutant could be rescued by brefeldin A treatment, which promotes the mixing of ER and Golgi membranes. Interestingly though, the palmitoylated mutant remained at the ER following brefeldin A washout and did not traffic to more distal membrane compartments. We propose that CSP has a weak membrane affinity that allows the protein to locate its partner Golgi-localized DHHC proteins directly by membrane "sampling." Mutations that enhance membrane association prevent sampling and lead to accumulation of CSP on cellular membranes such as the ER. The coupling of CSP palmitoylation to Golgi membranes may thus be an important requirement for subsequent sorting. 相似文献
14.
Protein S-acylation, more commonly known as protein palmitoylation, is a biological process defined by the covalent attachment of long chain fatty acids onto cysteine residues of a protein, effectively altering the local hydrophobicity and influencing its stability, localization and overall function. Observed ubiquitously in all eukaryotes, this post translational modification is mediated by the 23-member family of zDHHC protein acyltransferases in mammals. There are thousands of proteins that are S-acylated and multiple zDHHC enzymes can potentially act on a single substrate. Since its discovery, numerous methods have been developed for the identification of zDHHC substrates and the individual members of the family that catalyse their acylation. Despite these recent advances in assay development, there is a persistent gap in knowledge relating to zDHHC substrate specificity and recognition, that can only be thoroughly addressed through in vitro reconstitution. Herein, we will review the various methods currently available for reconstitution of protein S-acylation for the purposes of identifying enzyme–substrate pairs with a particular emphasis on the advantages and disadvantages of each approach. 相似文献
15.
A proline-rich region and nearby cysteine residues target XLalphas to the Golgi complex region
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XLalphas is a splice variant of the heterotrimeric G protein, Galpha(s), found on Golgi membranes in cells with regulated and constitutive secretion. We examined the role of the alternatively spliced amino terminus of XLalphas for Golgi targeting with the use of subcellular fractionation and fluorescence microscopy. XLalphas incorporated [(3)H]palmitate, and mutation of cysteines in a cysteine-rich region inhibited this incorporation and lessened membrane attachment. Deletion of a proline-rich region abolished Golgi localization of XLalphas without changing its membrane attachment. The proline-rich and cysteine-rich regions together were sufficient to target the green fluorescent protein, a cytosolic protein, to Golgi membranes. The membrane attachment and Golgi targeting of the fusion protein required the putative palmitoylation sites, the cysteine residues in the cysteine-rich region. Several peripheral membrane proteins found at the Golgi have proline-rich regions, including a Galpha(i2) splice variant, dynamin II, betaIII spectrin, comitin, and a Golgi SNARE, GS32. Our results suggest that proline-rich regions can be a Golgi-targeting signal for G protein alpha subunits and possibly for other peripheral membrane proteins as well. 相似文献
16.
Bastin G Singh K Dissanayake K Mighiu AS Nurmohamed A Heximer SP 《The Journal of biological chemistry》2012,287(34):28966-28974
Regulator of G-protein signaling (RGS) proteins are potent inhibitors of heterotrimeric G-protein signaling. RGS4 attenuates G-protein activity in several tissues. Previous work demonstrated that cysteine palmitoylation on residues in the amino-terminal (Cys-2 and Cys-12) and core domains (Cys-95) of RGS4 is important for protein stability, plasma membrane targeting, and GTPase activating function. To date Cys-2 has been the priority target for RGS4 regulation by palmitoylation based on its putative role in stabilizing the RGS4 protein. Here, we investigate differences in the contribution of Cys-2 and Cys-12 to the intracellular localization and function of RGS4. Inhibition of RGS4 palmitoylation with 2-bromopalmitate dramatically reduced its localization to the plasma membrane. Similarly, mutation of the RGS4 amphipathic helix (L23D) prevented membrane localization and its G(q) inhibitory function. Together, these data suggest that both RGS4 palmitoylation and the amphipathic helix domain are required for optimal plasma membrane targeting and function of RGS4. Mutation of Cys-12 decreased RGS4 membrane targeting to a similar extent as 2-bromopalmitate, resulting in complete loss of its G(q) inhibitory function. Mutation of Cys-2 did not impair plasma membrane targeting but did partially impair its function as a G(q) inhibitor. Comparison of the endosomal distribution pattern of wild type and mutant RGS4 proteins with TGN38 indicated that palmitoylation of these two cysteines contributes differentially to the intracellular trafficking of RGS4. These data show for the first time that Cys-2 and Cys-12 play markedly different roles in the regulation of RGS4 membrane localization, intracellular trafficking, and G(q) inhibitory function via mechanisms that are unrelated to RGS4 protein stabilization. 相似文献
17.
Targeting of tail‐anchored membrane proteins to subcellular organelles in Toxoplasma gondii
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Leah R. Padgett Gustavo Arrizabalaga William J. Sullivan Jr. 《Traffic (Copenhagen, Denmark)》2017,18(3):149-158
Proper protein localization is essential for critical cellular processes, including vesicle‐mediated transport and protein translocation. Tail‐anchored (TA) proteins are integrated into organellar membranes via the C‐terminus, orienting the N‐terminus towards the cytosol. Localization of TA proteins occurs posttranslationally and is governed by the C‐terminus, which contains the integral transmembrane domain (TMD) and targeting sequence. Targeting of TA proteins is dependent on the hydrophobicity of the TMD as well as the length and composition of flanking amino acid sequences. We previously identified an unusual homologue of elongator protein, Elp3, in the apicomplexan parasite Toxoplasma gondii as a TA protein targeting the outer mitochondrial membrane. We sought to gain further insight into TA proteins and their targeting mechanisms using this early‐branching eukaryote as a model. Our bioinformatics analysis uncovered 59 predicted TA proteins in Toxoplasma, 9 of which were selected for follow‐up analyses based on representative features. We identified novel TA proteins that traffic to specific organelles in Toxoplasma, including the parasite endoplasmic reticulum, mitochondrion, and Golgi apparatus. Domain swap experiments elucidated that targeting of TA proteins to these specific organelles was strongly influenced by the TMD sequence, including charge of the flanking C‐terminal sequence. 相似文献
18.
Blank M Wollberg J Gerlach F Reimann K Roesner A Hankeln T Fago A Weber RE Burmester T 《PloS one》2011,6(9):e25292
The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2)-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio) globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2) binding with a variable affinity (P(50)~1.3-12.5 torr), depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein. 相似文献
19.
The Yck2 protein is a plasma membrane-associated casein kinase 1 isoform that attaches to membranes via palmitoylation of its C terminus. We have demonstrated that Yck2p traffics to the plasma membrane on secretory vesicles. Because Akr1p, the palmitoyl transferase for Yck2p, is located on Golgi membranes, it is likely that Yck2p first associates with Golgi membranes, and then is somehow recruited to budding plasma membrane-destined vesicles. We show here that residues 499-546 are sufficient for minimal Yck2p palmitoylation and plasma membrane localization. We previously described normal plasma membrane targeting of a Yck2p construct with the final five amino acids of Ras2p substituting for the final two Cys residues of Yck2p. This Yck2p variant no longer requires Akr1p for membrane association, but targets normally. We have generated the C-terminal deletions previously shown to affect Yck2p membrane association in this variant to determine which residues are important for targeting and/or modification. We find that all of the sequences previously identified as important for plasma membrane association are required only for Akr1p-dependent modification. Furthermore, palmitoylation is sufficient for specific association of Yck2p with secretory vesicles destined for the plasma membrane. Finally, both C-terminal Cys residues are palmitoylated, and dual acylation is required for efficient membrane association. 相似文献
20.
A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles. 相似文献