The intracellular dynamic of protein palmitoylation

S-palmitoylation describes the reversible attachment of fatty acids (predominantly palmitate) onto cysteine residues via a labile thioester bond. This posttranslational modification impacts protein functionality by regulating membrane interactions, intracellular sorting, stability, and membrane micropatterning. Several recent findings have provided a tantalizing insight into the regulation and spatiotemporal dynamics of protein palmitoylation. In mammalian cells, the Golgi has emerged as a possible super-reaction center for the palmitoylation of peripheral membrane proteins, whereas palmitoylation reactions on post-Golgi compartments contribute to the regulation of specific substrates. In addition to palmitoylating and depalmitoylating enzymes, intracellular palmitoylation dynamics may also be controlled through interplay with distinct posttranslational modifications, such as phosphorylation and nitrosylation.     

Assays of protein palmitoylation

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.     

67-kD elastin-binding protein is a protective "companion" of extracellular insoluble elastin and intracellular tropoelastin

The 67-kD elastin-binding protein (EBP) mediates cell adhesion to elastin and elastin fiber assembly, and it is similar, if not identical, to the 67-kD enzymatically inactive, alternatively spliced beta-galactosidase. The latter contains an elastin binding domain (S- GAL) homologous both to the aorta EBP and to NH2-terminal sequences of serine proteinases (Hinek, A., M. Rabinovitch, F. W. Keeley, and J. Callahan. 1993. J. Clin. Invest. 91:1198-1205). We now confirm the functional importance of this homology by showing that elastolytic activity of a representative serine elastase, porcine pancreatic elastase, was prevented by an antibody (anti-S-GAL) and by competing with purified EBP or S-GAL peptide. Immunohistochemistry of adult aorta indicates that the EBP exists as a permanent component of mature elastic fibers. This observation, together with the in vitro studies, suggests that the EBP could protect insoluble elastin from extracellular proteolysis and contribute to the extraordinary stability of this protein. Double immunolabeling of fetal lamb aorta with anti-S- GAL and antitropoelastin antibodies demonstrated, under light and electron microscopy, intracellular colocalization of the proteins in smooth muscle cells (SMC). Incubation of SMC with galactosugars to dissociate tropoelastin from EBP caused intracellular aggregation of tropoelastin. A tropoelastin/EBP complex was extracted from SMC lysates by coimmunoprecipitation and cross-linking, and its functional significance was addressed by showing that its dissociation by galactosugars caused degradation of tropoelastin by endogenous serine proteinase(s). This suggests that the EBP may also serve as a "companion" to intracellular tropoelastin, protecting this highly hydrophobic protein from self-aggregation and proteolytic degradation.     

Formation and intracellular transport of a heterodimeric viral spike protein complex

We have analyzed the heterodimerization and intracellular transport from the ER to the Golgi complex (GC) of two membrane glycoproteins of a bunyavirus (Uukuniemi virus) that matures by a budding process in the GC. The glycoproteins G1 and G2, which form the viral spikes, are cotranslationally cleaved in the ER from a 110,000-D precursor. Newly synthesized G1 was transported to the GC and incorporated into virus particles about 30-45 min faster than newly synthesized G2. Analysis of the kinetics of intrachain disulfide bond formation showed that G1 acquired its mature form within 10 min, while completion of disulfide bond formation of G2 required a considerably longer time (up to 60 min). During the maturation process, G2 was transiently associated with the IgG heavy chain binding protein for a longer time than G1. Protein disulfide isomerase also coprecipitated with antibodies against G1 and G2. In virus particles, G1 and G2 were present exclusively as heterodimers. Immunoprecipitation with monoclonal antibodies showed that heterodimerization occurred rapidly, probably in the ER, between newly made G1 and mature, dimerization competent G2. Taken together, our results show that these two viral glycoproteins have different maturation kinetics in the ER. We conclude that the apparent different kinetics of ER to GC transport of G1 and G2 is due to the different rates by which these proteins fold and become competent to enter into heterodimeric complexes prior to exit from the ER.     

Prediction and analysis of protein palmitoylation sites

Palmitoylation is a universal and important lipid modification, involving a series of basic cellular processes, such as membrane trafficking, protein stability and protein aggregation. With the avalanche of new protein sequences generated in the post genomic era, it is highly desirable to develop computational methods for rapidly and effectively identifying the potential palmitoylation sites of uncharacterized proteins so as to timely provide useful information for revealing the mechanism of protein palmitoylation. By using the Incremental Feature Selection approach based on amino acid factors, conservation, disorder feature, and specific features of palmitoylation site, a new predictor named IFS-Palm was developed in this regard. The overall success rate thus achieved by jackknife test on a newly constructed benchmark dataset was 90.65%. It was shown via an in-depth analysis that palmitoylation was intimately correlated with the feature of the upstream residue directly adjacent to cysteine site as well as the conservation of amino acid cysteine. Meanwhile, the protein disorder region might also play an import role in the post-translational modification. These findings may provide useful insights for revealing the mechanisms of palmitoylation.     

A major 62-kD intranuclear matrix polypeptide is a component of metaphase chromosomes

We have isolated and partially characterized a major intranuclear matrix polypeptide from rat liver. This polypeptide, which is reversibly stabilized into the intranuclear matrix under conditions which promote intermolecular disulfide bond formation, has a Mr of 62,000 and pI of 6.8-7.2 as determined by two-dimensional IEF/SDS-PAGE. A chicken polyclonal antiserum was raised against the polypeptide purified from two-dimensional polyacrylamide gels. Affinity-purified anti-62-kD IgG was prepared and used to immunolocalize this polypeptide in rat liver tissue hepatocytes. In interphase hepatocytes the 62-kD antigen is localized in small, discrete patches within the nucleus consistent with the distribution of chromatin. The staining is most prominent at the nuclear periphery and somewhat less dense in the nuclear interior. Nucleoli and cytoplasm are devoid of staining. During mitosis the 62-kD antigen localizes to the condensed chromosomes with no apparent staining of cytoplasmic areas. The chromosomal staining during mitosis is uniform with no suggestion of the patching seen in interphase nuclei. Fractionation and immunoblotting studies using rat hepatoma tissue culture cells blocked in metaphase with colcemid confirm the chromosomal localization of this 62-kD intranuclear protein during mitosis. The 62-kD polypeptide fractionates completely with metaphase chromosome scaffolds generated by sequential treatment of isolated chromosomes with DNAse I and 1.6 M NaCl, suggesting that this major 62-kD intranuclear protein may be involved in maintaining metaphase chromosomal architecture.     

Recovery from photoinhibition: effect of light and inhibition of protein synthesis of 32-kD chloroplast protein

Recovery from photoinhibition of photosynthesis in intact Lemna gibba was studied in presence of the protein synthesis inhibitors chloramphenicol and cycloheximide. Exposure to an irradiance of 1000 mol m^-2s^-1 in N₂ for 90 min induced 80% photoinhibition. The plants recovered photosynthesis when transfered to normal irradiances (210 mol m^-2s^-1) and air. Chloramphenicol added to the medium was taken up by the plant and reduced photosynthesis slightly. Recovery from photoinhibition was more inhibited than photosynthesis. Cycloheximide was also taken up by the plants and reduced synthesis of light harvesting chlorophyll protein: however, neither photosynthesis nor recovery were much affected. Synthesis of 32-kD chloroplast protein during recovery was inhibited by chloramphenicol, but not by cycloheximide. Synthesis of 32-kD protein was enhanced by 20–210 mol m^-2s^-1 light. The results support the hypothesis that synthesis of 32-kD protein is important for recovery of photosynthesis after photoinhibition.     

Effect of 2-fluoropalmitate,cerulenin and tunicamycin on the palmitoylation and intracellular translocation of myelin proteolipid protein

We have investigated the effect of documented protein palmitoylation inhibitors on the fatty acylation and intracellular transport of myelin proteolipid protein (PLP). To this end, brain slices from 20-day-old rats were incubated with either [³H]palmitate or [³H]leucine in the presence or absence of various concentrations of 2-fluoropalmitate (FP), cerulenin (CER), or tunicamycin (TM). FP ( 10 M) decreased the cellular uptake of [³H]palmitate and consequently reduced the labeling of palmitoyl-CoA, glycerolipids and PLP. CER ( 1 mM) reduced the palmitoylation of PLP with a concomitant decline in protein thiols. Consistent with being a fatty acyl-CoA analogue, TM ( 200 M) diminished the palmitoylation of PLP and lipids while increasing the amount of [³H]palmitoyl-CoA. Although both CER and TM decreased protein palmitoylation, only the latter affected the appearance of newly synthesized PLP into myelin. Because TM, but not CER, also reduced the formation of lipids, it is concluded that palmitoylation is not required for intracellular transport. Finally, comparison of the effect of TM in brain slices and in a cell-free system suggests that palmitoylation of PLP in whole cells may be an enzymatic process.     

Labeling and quantifying sites of protein palmitoylation

As a reversible posttranslational modification, protein palmitoylation has the potential to regulate the trafficking and function of a variety of proteins. However, the extent, function, and dynamic nature of palmitoylation are poorly resolved because of limitations in assay methods. Here, we introduce methods where hydroxylamine-mediated cleavage of the palmitoyl-thioester bond generates a free sulfhydryl, which can then be specifically labeled with sulfhydryl-reactive reagents. This methodology is more sensitive and allows for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or nonradioactive, and can be used to assay the palmitoylation of proteins expressed in vivo in brain or other tissues.     

蛋白质的棕榈酰化修饰

棕榈酰化修饰是蛋白质翻译后脂质修饰的重要形式,是调控蛋白质的转运、稳定、定位和功能的重要机制,同时,棕榈酰化修饰还参与多种细胞生物学进程,与许多疾病的发生发展密切相关。本文主要就蛋白质棕榈酰化及其修饰酶与蛋白质功能、相关疾病的关系做一综述。     

On the mechanism of protein palmitoylation

Protein palmitoylation or, more specifically, S-acylation is a reversible post-translational lipid modification. Despite the identification of several proteins that are altered in this way, our understanding of the enzymology of this process has been hampered by the lack of well-characterized acyltransferases. We now know of three proteins in Saccharomyces cerevisiae that promote palmitoylation: effector of Ras function (Erf2), ankyrin-repeat-containing protein (Akr1) and the SNARE protein Ykt6. Erf2 and Akr1 are integral membrane proteins that contain a cysteine-rich domain and an Asp-His-His-Cys motif, both of which catalyse acylation at the carboxyl terminus of their target proteins. Recently, we discovered that Ykt6 mediates the amino-terminal acylation of the fusion protein Vac8. Even though these three proteins differ in sequence, topology, size and substrate specificity, they might function in a similar manner. In this review, we discuss these observations in the context of a potential general mechanism of acylation.     

VIP21, a 21-kD membrane protein is an integral component of trans-Golgi- network-derived transport vesicles

In simple epithelial cells, apical and basolateral proteins are sorted into separate vesicular carriers before delivery to the appropriate plasma membrane domains. To dissect the putative sorting machinery, we have solubilized Golgi-derived transport vesicles with the detergent CHAPS and shown that an apical marker, influenza haemagglutinin (HA), formed a large complex together with several integral membrane proteins. Remarkably, a similar set of CHAPS-insoluble proteins was found after solubilization of a total cellular membrane fraction. This allowed the cloning of a cDNA encoding one protein of this complex, VIP21 (Vesicular Integral-membrane Protein of 21 kD). The transiently expressed protein appeared on the Golgi-apparatus, the plasma membrane and vesicular structures. We propose that VIP21 is a component of the molecular machinery of vesicular transport.     

The on-off story of protein palmitoylation

Palmitoylation is one of the most frequent post-translational modifications found on proteins. It contributes to membrane association, protein sorting and many other processes. Through its reversibility, palmitoylation also provides mechanisms to regulate the functional activities of integral and peripheral membrane proteins. Here we discuss evidence that proteins can be palmitoylated at different locations in the cell, how targeting to these locations might be directed, and aspects of the proposed functions of palmitoylation.     

Global profiling of dynamic protein palmitoylation

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.     

The fat controller: roles of palmitoylation in intracellular protein trafficking and targeting to membrane microdomains (Review)

The attachment of palmitic acid to the amino acid cysteine via thioester linkage (S-palmitoylation) is a common post-translational modification of eukaryotic proteins. In this review, we discuss the role of palmitoylation as a versatile protein sorting signal, regulating protein trafficking between distinct intracellular compartments and the micro-localization of proteins within membranes.     

Specificity of transmembrane protein palmitoylation in yeast

Many proteins are modified after their synthesis, by the addition of a lipid molecule to one or more cysteine residues, through a thioester bond. This modification is called S-acylation, and more commonly palmitoylation. This reaction is carried out by a family of enzymes, called palmitoyltransferases (PATs), characterized by the presence of a conserved 50- aminoacids domain called "Asp-His-His-Cys- Cysteine Rich Domain" (DHHC-CRD). There are 7 members of this family in the yeast Saccharomyces cerevisiae, and each of these proteins is thought to be responsible for the palmitoylation of a subset of substrates. Substrate specificity of PATs, however, is not yet fully understood. Several yeast PATs seem to have overlapping specificity, and it has been proposed that the machinery responsible for palmitoylating peripheral membrane proteins in mammalian cells, lacks specificity altogether.Here we investigate the specificity of transmembrane protein palmitoylation in S. cerevisiae, which is carried out predominantly by two PATs, Swf1 and Pfa4. We show that palmitoylation of transmembrane substrates requires dedicated PATs, since other yeast PATs are mostly unable to perform Swf1 or Pfa4 functions, even when overexpressed. Furthermore, we find that Swf1 is highly specific for its substrates, as it is unable to substitute for other PATs. To identify where Swf1 specificity lies, we carried out a bioinformatics survey to identify amino acids responsible for the determination of specificity or Specificity Determination Positions (SDPs) and showed experimentally, that mutation of the two best SDP candidates, A145 and K148, results in complete and partial loss of function, respectively. These residues are located within the conserved catalytic DHHC domain suggesting that it could also be involved in the determination of specificity. Finally, we show that modifying the position of the cysteines in Tlg1, a Swf1 substrate, results in lack of palmitoylation, as expected for a highly specific enzymatic reaction.     

Global analysis of protein palmitoylation in yeast

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.     

Endocytosis, intracellular transport and transcytosis of the toxic protein ricin by a polarized epithelium

The toxic plant protein ricin binds to both the apical and basolateral surface domains of MDCK (strain I) cells grown on polycarbonate filters. Endocytosis of 125I-labeled ricin was not only higher from the basolateral than from the apical surface--an observation which can be explained by the higher surface area of the basolateral surface--but it also appeared to be more efficient when measured as a percentage of total cell-associated ricin. Monovalent ricin-horseradish peroxidase (Ri-HRP), which is known to behave like native ricin with respect to intracellular transport, also binds to, and is taken up from, both the apical and the basolateral surfaces. Initially, after 10 to 15 min, molecules taken up from the two surface domains at 37 degrees C are present in two separate (basolateral and apical) early endosomal populations. This can also be obtained by incubating for 60 min at 18 degrees C. However, after 30 to 60 min at 37 degrees C, most internalized ligand is found in apical lysosomes, regardless from which surface endocytosis took place. Experiments with endocytosis of cationized ferritin from the apical pole and HRP or Ri-HRP from the basolateral pole showed that intermixing in apical lysosomes (or prelysosomes) of molecules taken up from the two poles occurs. Bidirectional transcytosis involving coated pits of both 125I-labeled ricin and Ri-HRP was demonstrated and was found to be most efficient (as measured in per cent of endocytosed toxin) from the apical pole. Transcytosis was strongly reduced at 18 degrees C, and no transepithelial transport of ricin could be measured at 4 degrees C. Transcytosed ricin was intact and could intoxicate new cells. Finally, delivery of ricin internalized from both the apical and the basolateral surface to the apically localized trans-Golgi network occurred at 37 degrees C but not at 18 degrees C, and ricin inhibited protein synthesis largely with the same kinetics following uptake from the two poles. Incubation at 18 degrees C strongly inhibited the toxic effect of ricin. These data show that ricin can intoxicate epithelia from both sides and also penetrate tight epithelial barriers in intact form.     

Isolation and partial characterization of a 110-kD dimer actin-binding protein

Two Triton-insoluble fractions were isolated from Acanthamoeba castellanii. The major non-membrane proteins in both fractions were actin (30-40%), myosin II (4-9%), myosin I (1-5%), and a 55-kD polypeptide (10%). The 55-kD polypeptide did not react with antibodies against tubulins from turkey brain, paramecium, or yeast. All of these proteins were much more concentrated in the Triton-insoluble fractions than in the whole homogenate or soluble supernatant. The 55-kD polypeptide was extracted with 0.3 M NaCl, fractionated by ammonium sulfate, and purified to near homogeneity by DEAE-cellulose and hydroxyapatite chromatography. The purified protein had a molecular mass of 110 kD and appeared to be a homodimer by isoelectric focusing. The 110-kD dimer bound to F-actin with a maximal binding stoichiometry of 0.5 mol/mol of actin (1 mol of 55-kD subunit/mol of actin). Although the 110-kD protein enhanced the sedimentation of F-actin, it did not affect the low shear viscosity of F-actin solutions nor was bundling of F-actin observed by electron microscopy. The 110-kD dimer protein inhibited the actin-activated Mg2+-ATPase activities of Acanthamoeba myosin I and myosin II in a concentration-dependent manner. By indirect immunofluorescence, the 110-kD protein was found to be localized in the peripheral cytoplasm near the plasma membrane which is also enriched in F-actin filaments and myosin I.