Posttranslational membrane attachment and dynamic fatty acylation of a neuronal growth cone protein, GAP-43

Growth cones, the motile apparatus at the ends of elongating axons, are sites of extensive and dynamic membrane-cytoskeletal interaction and insertion of new membrane into the growing axon. One of the most abundant proteins in growth cone membranes is a protein designated GAP-43, whose synthesis increases dramatically in most neurons during periods of axon development or regeneration. We have begun to explore the role of GAP-43 in growth cone membrane functions by asking how the protein interacts with those membranes. Membrane-washing experiments indicate that mature GAP-43 is tightly bound to growth cone membranes, and partitioning of Triton X-114-solubilized GAP-43 between detergent-enriched and detergent-depleted phases indicates considerable hydrophobicity. The hydrophobic behavior of the protein is modulated by divalent cations, particularly zinc and calcium. In vivo labeling of GAP-43 in neonatal rat brain with [35S]methionine shows that GAP-43 is initially synthesized as a soluble protein that becomes attached to membranes posttranslationally. In tissue culture, both rat cerebral cortex cells and neuron-like PC12 cells actively incorporate [3H]palmitic acid into GAP-43. Isolated growth cones detached from their cell bodies also incorporate labeled fatty acid into GAP-43, suggesting active turnover of the fatty acid moieties on the mature protein. Hydrolysis of ester-like bonds with neutral hydroxylamine removes the bound fatty acid and exposes new thiol groups on GAP-43, suggesting that fatty acid is attached to the protein's only two cysteine residues, located in a short hydrophobic domain at the amino terminus. Modulation of the protein's hydrophobic behavior by divalent cations suggests that other domains, containing large numbers of negatively charged residues, might also contribute to GAP-43-membrane interactions. Our observations suggest a dynamic and reversible interaction of GAP-43 with growth cone membranes.     

Identification of a novel protein (G210) specific to the Golgi apparatus

A monoclonal antibody, 3C9, has enabled the detection of a novel Golgi-specific protein in bovine tissues. Immunohistochemical studies at the light microscopic level have detected the 3C9 antigen only in certain cells: exocrine pancreas, gut epithelium, and thymus epithelium. Examination of gut and pancreas by immunoelectron microscopy showed a localization exclusive to the Golgi apparatus. The relative molecular weight of the antigen detected by immunoblotting is 210,000 daltons. The antigen is not extracted from microsomal membranes of bovine gut epithelium by sodium carbonate solutions. Furthermore, the 3C9 antigen enters into the detergent phase when Triton X-114 partitioning methods are used. These data strongly suggest that this novel antigen is an intrinsic membrane protein, resident in the Golgi apparatus of certain cells. Moreover, they enhance the hypothesis that the distribution of enzymes and polypeptides in the Golgi apparatus is cell specific.     

Different oligosaccharide processing of the membrane-integrated and the secretory form of gp 80 in rat liver.

Rat liver synthesizes a glycoprotein with Mr of 80.000 (gp 80) which is partly inserted into the plasma membrane and partly secreted into the serum. The membrane-integrated and the secretory form of this glycoprotein have an identical peptide pattern, but different N-linked glycans. Whereas gp 80 from the serum is glycosylated with complex-type oligosaccharides, gp 80 from the plasma membrane has high mannose glycans. Phase separation with Triton X-114 showed that membrane-integrated gp 80 contains hydrophobic portions, whereas secretory gp 80 has hydrophilic properties. Intracellular transport and oligosaccharide processing of gp 80 were studied in vivo in the endoplasmic reticulum, the Golgi apparatus and plasma membranes of rat liver and in serum using pulse-chase labeling with L-[35S]methionine and immunoprecipitation. Peak labeling of gp 80 was reached in the endoplasmic reticulum 10 min after the pulse, in the Golgi apparatus 20 min later, and in the plasma membrane after 2 h; in the serum the specific radioactivity was steadily increasing during the experiment. Gp 80 of the endoplasmic reticulum was completely sensitive to endo-beta-N-glucosaminidase H (endo H), but simultaneously occurred in the Golgi apparatus in an endo H-sensitive and endo H-resistant form. The endo H-sensitive form was transported to the plasma membrane, the endo H-resistant species secreted into the serum. Conversion from the endo H-sensitive to the endo H-resistant form was completed within 10 min after transfer of gp 80 to the Golgi apparatus.(ABSTRACT TRUNCATED AT 250 WORDS)     

G-proteins of rat liver membranes. Subcellular compartmentation and disposition in the plasma membrane

Summary The distribution of the alpha- and beta-subunits of G-proteins and their disposition in rat liver plasma and intracellular membranes was investigated. Western blotting, using antibodies that recognised the alpha-subunit of the inhibitory and the beta-subunits of most G-proteins, identified 41 and 36 kDa polypeptides respectively in all plasma membrane functional domains, in endosomes as well as in Golgi membranes. Lysosomes were devoid of these subunits. The highest levels of G-protein subunits were found in bile canalicular plasma membranes prepared by density gradient centrifugation followed by free-flow electrophoresis. Separation of membrane proteins into extrinsic and intrinsic components was carried out by extraction of the membranes at pH 11.0 and by partitioning the membranes in Triton X-114/aqueous phases. The results demonstrated that the alpha- and beta-subunits were tightly associated with the hepatic membranes but they could be solubilised by extraction with detergent, e.g. SDS. Prolonged incubation in the presence of GTP analogues also released up to approximately 50% of the alpha-subunit of inhibitory G-proteins from membranes. The beta-subunit was still associated with membranes after alkaline extraction. The results emphasise the strong association of G-protein subunits with liver membranes, and show that these proteins are distributed widely in the plasma membrane and along the endocytic pathways of hepatocytes.     

Two membrane-bound forms of tyrosylprotein sulfotransferase as revealed by phase partitioning in Triton X-114.

Tyrosylprotein sulfotransferase (TPST) is a membrane-associated enzyme of the trans Golgi network that catalyzes the posttranslational sulfation of a variety of secretory and membrane proteins. We have analyzed the membrane association of TPST in Golgi-enriched fractions from bovine adrenal medulla using carbonate treatment (pH 11) and Triton X-114 phase partitioning. TPST was not extracted by carbonate. Triton X-114 phase partitioning revealed that, unexpectedly, TPST from non-carbonate-treated membranes was present in both, a hydrophilic and a hydrophobic form with apparent sedimentation coefficients of approximately 13 and approximately 6, respectively. Extraction of membranes with carbonate converted the hydrophilic form TPST to the hydrophobic form. Addition of the carbonate extract to TPST solubilized from carbonate-treated membranes converted the hydrophobic form of the enzyme to the hydrophilic form. This conversion of TPST was specific in that it was not observed for the bulk of the proteins present in the carbonate-treated membranes. The factor in the carbonate extract responsible for this conversion, referred to as "phase-transfer factor", (i) was precipitable with ammonium sulfate and polyethylene glycol, (ii) was non-dialyzable, (iii) was not extracted from membranes by 0.5 M NaCl, and (iv) appeared to be more abundant than TPST itself. These results show that TPST is an integral membrane protein and suggested that the enzyme may exist in a complex with a peripheral membrane protein. Moreover, a phase-transfer factor was also observed in another system, PC12 cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP- sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.     

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.     

Cell-free transfer and sorting of membrane lipids in spinach

Summary The donor and acceptor specificity of cell-free transfer of radiolabeled membrane constituents, chiefly lipids, was examined using purified fractions of endoplasmic reticulum, Golgi apparatus, nuclei, plasma membrane, tonoplast, mitochondria, and chloroplasts prepared from green leaves of spinach. Donor membranes were radiolabeled with [¹⁴C]acetate. Acceptor membranes were unlabeled and immobilized on nitrocellulose filters. The assay was designed to measure membrane transfer resulting from ATP-and temperature-dependent formation of transfer vesicles by the donor fraction in solution and subsequent attachment and/or fusion of the transfer vesicles with the immobilized acceptor. When applied to the analysis of spinach fractions, significant ATP-dependent transfer in the presence of cytosol was observed only with endoplasmic reticulum as donor and Golgi apparatus as acceptor. Transfer in the reverse direction, from Golgi apparatus to endoplasmic reticulum, was only 0.2 to 0.3 that from endoplasmic reticulum to Golgi apparatus. ATP-dependent transfers also were indicated between nuclei and Golgi apparatus from regression analysis of transfer kinetics. Specific transfer between Golgi apparatus and plasma membrane and, to a lesser extent, from plasma membrane to Golgi apparatus was observed at 25°C compared to 4°C but was not ATP plus cytosol-dependent. All other combinations of organelles and membranes exhibited no ATP plus cytosol-dependent transfer and only small increments of specific transfer comparing transfer at 37°C to transfer at 4°C. Thus, the only combinations of membranes capable of significant cell-free transfer in vitro were those observed by electron microscopy of cells and tissues to be involved in vesicular transport in vivo (endoplasmic reticulum, Golgi apparatus, plasma membrane, nuclear envelope). Of these, only with endoplasmic reticulum (or nuclear envelope) and Golgi apparatus, where transfer in situ is via 50 to 70 nm transition vesicles, was temperature-and ATP-dependent transfer of acetatelabeled membrane reproduced in vitro. Lipids transferred included phospholipids, mono-and diacylglycerols, and sterols but not triacylglycerols or steryl esters, raising the possibility of lipid sorting or processing to exclude transfer of triacylglycerols and steryl esters at the endoplasmic reticulum to Golgi apparatus step.     

Membrane lipids of rat liver lysosomes prepared by freeflow electrophoresis

Rat liver lysosomes were isolated by free-flow electrophoresis and were examined morphologically and enzymatically for purity. Their membrane fraction was prepared by osmotic shock and analyzed for cholesterol, phospholipids and fatty acids. The results were compared with the membrane fraction of Triton WR 1339-filled lysosomes and with mitochondria. The cholesterol content (0.269 M cholesterol per M lipid phosphorus), the sphingomyelin concentration (7.9% of total lipid phosphorus) and the degree of unsaturation of fatty acids (38–45%) were found to be intermediate between those of membranes of Triton WR 1339-filled lysosomes (“plasma membrane-like”) and mitochondria (“endoplasmic reticulum-like”). The similarity of these results with corresponding data for the Golgi apparatus support the present view concerning the formation of primary lysosomes via the Golgi apparatus. The drastic changes in the lipid composition found after overloading with Triton WR 1339 confirm that the plasma membrane participates in the formation of the secondary lysosomal membrane. The data presented here underline the significance of the analysis of membrane lipids in evaluating correlations between morphologically different but functionally closely related membrane types.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea.

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly puridied nuclei exhibited specific binding with 125I-labeled human choriogonadotropin, [3H]prostaglandin E1 and [3H]prostaglandin F2 alpha. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E1) and 8.9% (prostaglandin F2 alpha) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F2 alpha binding site and 5'-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21--26% (prostaglandins) of original specific binding despite virtual disappearance of 5'-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound 125I-labeled human choriogonadotropin and [3H]prostaglandin F2 alpha to the same extent or significantly more ([3H]prostaglandin E1, P less than 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

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.     

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.     

The Autodepalmitoylating Activity of APT Maintains the Spatial Organization of Palmitoylated Membrane Proteins

The localization and signaling of S-palmitoylated peripheral membrane proteins is sustained by an acylation cycle in which acyl protein thioesterases (APTs) depalmitoylate mislocalized palmitoylated proteins on endomembranes. However, the APTs are themselves reversibly S-palmitoylated, which localizes thioesterase activity to the site of the antagonistc palmitoylation activity on the Golgi. Here, we resolve this conundrum by showing that palmitoylation of APTs is labile due to autodepalmitoylation, creating two interconverting thioesterase pools: palmitoylated APT on the Golgi and depalmitoylated APT in the cytoplasm, with distinct functionality. By imaging APT-substrate catalytic intermediates, we show that it is the depalmitoylated soluble APT pool that depalmitoylates substrates on all membranes in the cell, thereby establishing its function as release factor of mislocalized palmitoylated proteins in the acylation cycle. The autodepalmitoylating activity on the Golgi constitutes a homeostatic regulation mechanism of APT levels at the Golgi that ensures robust partitioning of APT substrates between the plasma membrane and the Golgi.     

The catalytic site of the pectin biosynthetic enzyme alpha-1,4-galacturonosyltransferase is located in the lumen of the Golgi

Alpha-1,4-galacturonosyltransferase (GalAT) is an enzyme required for the biosynthesis of the plant cell wall pectic polysaccharide homogalacturonan (HGA). GalAT activity in homogenates from pea (Pisum sativum L. var. Alaska) stem internodes co-localized in linear and discontinuous sucrose gradients with latent UDPase activity, an enzyme marker specific for Golgi membranes. GalAT activity was separated from antimycin A-insensitive NADH:cytochrome c reductase and cytochrome c oxidase activities, enzyme markers for the endoplasmic reticulum and the mitochondria, respectively. GalAT and latent UDPase activities were separated from the majority (80%) of callose synthase activity, a marker for the plasma membrane, suggesting that little or no GalAT is present in the plasma membrane. GalAT activities in proteinase K-treated and untreated Golgi vesicles were similar, whereas no GalAT activity was detected after treating Golgi vesicles with proteinase K in the presence of Triton X-100. These results demonstrate that the catalytic site of GalAT resides within the lumen of the Golgi. The products generated by Golgi-localized GalAT were converted by endopolygalacturonase treatment to mono- and di-galacturonic acid, thereby showing that GalAT synthesizes 1-->4-linked alpha-D-galacturonan. Our data provide the first enzymatic evidence that a glycosyltransferase involved in HGA synthesis is present in the Golgi apparatus. Together with prior results of in vivo labeling and immunocytochemical studies, these results show that pectin biosynthesis occurs in the Golgi. A model for the biosynthesis of the pectic polysaccharide HGA is proposed.     

Double Triton X-114 Phase Partitioning for the Purification of Plant Cytochromes P450 and Removal of Green Pigments

A double Triton X-114 phase partitioning procedure that separates plant cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase partitioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partitionings were carried out using phosphate buffer. We found that the partitioning of the cytochromes P450 could be shifted to a pigment-free Triton X-114 poor phase by changing the buffer component to borate. The protein extract containing the cytochromes P450 but devoid of green pigment was subjected to a second phase partitioning step before which the buffer was changed from borate to phosphate. This second phase partitioning step produced a Triton X-114-rich phase highly enriched in cytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages of purification using column chromatography. The double phase partitioning procedure takes 3–4 h to complete. This is very fast compared to traditional purification schemes for cytochromes P450 which involve multiple of column chromatographic steps. Plant cytochromes P450 are labile, low abundant proteins that are difficult to isolate. The double Triton X-114 phase partitioning here reported thus constitutes a versatile, efficient purification procedure circumventing many of the problems previously encountered.     

Akr1p-dependent palmitoylation of Yck2p yeast casein kinase 1 is necessary and sufficient for plasma membrane targeting

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.     

Further studies on the role of phospholipids in determining the characteristics of mitochondrial binding sites for type I hexokinase

Previous work has indicated that two types (A and B) of binding sites for hexokinase exist, but in different proportions, on brain mitochondria from various species. Hexokinase is readily solubilized from Type A sites by glucose 6-phosphate (Glc-6-P), while hexokinase bound to Type B sites remains bound even in the presence of Glc-6-P. Type A:Type B ratios are approximately 90:10, 60:40, 40:60, and 20:80 for brain mitochondria from rat, rabbit, bovine and human brain, respectively. The present study has indicated that MgCl2-dependent partitioning of mitochondrially bound hexokinase into a hydrophobic (Triton X-114) phase is generally correlated with the proportion of Type B sites. This partitioning behavior is sensitive to phospholipase C, implying that the factor(s) responsible for conferring hydrophobic character is(are) phospholipid(s). Substantial differences were also seen in the resistance of hexokinase, bound to brain mitochondria from various species, to solubilization by Triton X-100, Triton X-114, or digitonin. This resistance increased with proportion of Type B sites. Enrichment of bovine brain mitochondria in acidic phospholipids (phosphatidylserine or phosphatidylinositol), but not phosphatidylcholine or phosphatidylethanolamine, substantially increased solubilization of the enzyme after incubation at 37 degrees C. Collectively, the results imply that the Type A and Type B sites are located in membrane domains of different lipid composition, the Type A sites being in domains enriched in acidic phospholipids which lead to greater susceptibility to solubilisation by Glc-6-P.     

Palmitoylation of p59fyn is reversible and sufficient for plasma membrane association.

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.     

Subcellular localization and topology of homogalacturonan methyltransferase in suspension-cultured Nicotiana tabacum cells

Pectin is a complex polysaccharide in the primary walls of all plant cells that is thought to be synthesized in the cellular endomembrane system and inserted into the wall via exocytosis. The most abundant pectic polysaccharide, homogalacturonan, is partially methylesterified within the cell by the pectin methyltransferase homogalacturonan methyltransferase (HGA-MT). The subcellular location of HGA-MT activity was determined in tobacco (Nicotiana tabacum L. cv. Samsun) cell membranes separated on linear sucrose gradients. The activity of HGA-MT and two enzymatic markers of the Golgi apparatus, IDPase and UDPase, were found to be located in the same membrane fraction. No NADH cytochrome c reductase activity, a marker for the endoplasmic reticulum, was detected in the Golgi fraction. Homogalacturonan methyltransferase activity was not reduced by protease treatment of intact membranes or membranes treated with 0.01% Triton X-100. In contrast, HGA-MT activity was reduced by protease treatment of membranes permeabilized with 0.02% Triton X-100. The sensitivity of HGA-MT in detergent-permeabilized membranes, and the lack of inhibition of HGA-MT activity by protease-treatment of intact membranes, provides evidence that the catalytic site of HGA-MT is located on the lumenal side of the Golgi. Received: 2 December 1998 / Accepted: 9 February 1999