Low Molecular Mass GTP-binding Proteins Research Articles

Expression of Ras homologous B protein in the human scalp skin and hair follicles: hair follicle cycle stages‐associated changes

RhoB belongs to the Ras homologous (Rho) subfamily which consists of low molecular weight mass GTP-binding proteins. Rho proteins are regulatory molecules that mediate changes in cell shape, contractility, motility and gene expression. To test the hypothesis that 'RhoB protein is expressed in the human skin and its expression undergoes hair follicle cycle dependent changes'. To test this hypothesis, we examined the expression of RhoB in the normal human skin and hair follicles (HFs) using immunohistochemical methods. A total of 50 normal human scalp skin specimens were obtained from 50 females (age: 53-57 years) undergoing elective cosmetic plastic surgery. The specimens were obtained from both frontal and temporal regions of the scalp. A total of 50 HF, (35 anagen, 10 catagen and 5 telogen) were examined in each case using immunohistologic staining methods. Semiquantitative analysis was done. RhoB protein was strongly expressed in the various elements of the human scalp skin and hair follicles. In the epidermis, a moderate RhoB immunoreactivity was found in all layers except stratum corneum where RhoB protein was completely absent. In sebaceous glands, a strong RhoB immunoreactivity was detected in all sebaceocytes. In the hair follicles, the expression of RhoB protein showed hair follicle cycle stages-associated changes, i.e. strong expression during anagen, but weak and completely absent expressions during catagen and telogen phases, respectively. Semiquantitative analysis revealed statistically significant high expression values (staining intensity, percentage of positive cells and immunoreactivity scores) in the anagen VI hair follicles compared to either cantagen or telogen ones (p < 0.05). Similarly, RhoB protein expression was significantly high in the stratum basale, stratum spinosum and sebaceous glands compared to stratum granulosum (p < 0.05). Here we report, for the first time, the distribution of RhoB protein in the human scalp skin and hair follicles. We also provide the first indication that there are variations in the expression of this protein in the different stages of the hair cycle.

Clostridium difficile toxins: More than mere inhibitors of Rho proteins

Toxin A (TcdA) and Toxin B (TcdB) are the major pathogenicity factors of the Clostridium difficile-associated diarrhoea (CDAD). The single-chained protein toxins enter their target cells by receptor-mediated endocytosis. New data show the critical role of auto-catalytic processing for target cell entry. Inside the cell, the toxins mono-glucosylate and thereby inactivate low molecular mass GTP-binding proteins of the Rho subfamily. Toxin-treated cells respond to RhoA glucosylation with up-regulation and activation of the pro-apoptotic Rho family protein RhoB. These data reinforce the critical role of the glucosyltransferase activity for programmed cell death and show that TcdA and TcdB, generally classified as broad-spectrum inhibitors of Rho proteins, are also capable of activating Rho proteins.

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

The family of C3-like exoenzymes comprises seven bacterial ADP-ribosyltransferases of different origin. The common hallmark of these exoenzymes is the selective N-ADP-ribosylation of the low molecular mass GTP-binding proteins RhoA, B, and C and inhibition of signal pathways controlled by Rho GTPases. Therefore, C3-like exoenzymes were applied as pharmacological tools for analyses of cellular functions of Rho protein in numerous studies. Recent structural and functional analyses of C3-like exoenzymes provide detailed information on the molecular mechanisms and functional consequences of ADP-ribosylation catalyzed by these toxins. More recently additional non-enzymatic actions of C3-like ADP-ribosyltransferases have been identified showing that C3 transferases from Clostridium botulinum and Clostridium limosum form a GDI-like complex with the Ras-like low molecular mass GTPase Ral without ADP-ribosylation. These results add novel information on the molecular mode of action(s) of C3-like exoenzymes and are discussed in this review.

Organellar Proteomics

The zymogen granule (ZG) is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and has been a model for studying secretory granule functions. In an initial effort to comprehensively understand the functions of this organelle, we conducted a proteomic study to identify proteins from highly purified ZG membranes. By combining two-dimensional gel electrophoresis and two-dimensional LC with tandem mass spectrometry, 101 proteins were identified from purified ZG membranes including 28 known ZG proteins and 73 previously unknown proteins, including SNAP29, Rab27B, Rab11A, Rab6, Rap1, and myosin Vc. Moreover several hypothetical proteins were identified that represent potential novel proteins. The ZG localization of nine of these proteins was further confirmed by immunocytochemistry. To distinguish intrinsic membrane proteins from soluble and peripheral membrane proteins, a quantitative proteomic strategy was used to measure the enrichment of intrinsic membrane proteins through the purification process. The iTRAQ ratios correlated well with known or Transmembrane Hidden Markov Model-predicted soluble or membrane proteins. By combining subcellular fractionation with high resolution separation and comprehensive identification of proteins, we have begun to elucidate zymogen granule functions through proteomic and subsequent functional analysis of its membrane components.

Identification of the catalytic site of clostridial ADP-ribosyltransferases.

The catalytic sites of clostridial ADP-ribosyltransferases were studied by photoaffinity-labelling with [carbonyl-14C]NAD+. In C3-like transferases, which are known to modify low molecular mass GTP-binding Rho proteins, Glu-174 was identified to be essential for catalysis. In C. perfringens iota toxin, Glu-380 and Glu-378 may have pivotal roles in the active site of this actin-ADP-ribosylating toxin.

Association ofRalGTP-Binding Protein with Human Platelet Dense Granules

GTP-binding proteins of molecular mass of 24–27 kDa were detected in the dense granule fraction of human platelets when nitrocellulose blots containing proteins separated by SDS-polyacrylamide gel electrophoresis were incubated with [α-32P]GTP. Further analysis, using isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis, resolved the dense granule 27 kDa and 24 kDa GTP-binding proteins into four distinct forms each. GTP-binding proteins in the total particulate fraction were resolved into seven 27 kDa and four 24 kDa forms. Immunoblotting with antiserum against known platelet low molecular mass GTP-binding proteins demonstrated thatrap2 andG25K/CDC42Hs proteins, although present in platelets, were not detected in the dense granule fraction. However,ralwas one of the proteins associated with dense granules. Association of specific low molecular mass GTP-binding proteins with dense granules suggests a potential role for these proteins in regulating the release of storage contents from this granule.

Expression of a 24 kDa GTP-binding protein (Gn24) is increased in lovastatin treated human erythroleukemia cells.

A major 27 kDa particulate and a minor 24 kDa cytosolic GTP-binding protein was detected in HEL cells upon incubation with [alpha-32P]GTP of nitrocellulose blots containing polypeptides separated using SDS-PAGE. Addition of lovastatin (30 microM) to HEL cells in culture inhibited protein synthesis by approximately 35%. However, this treatment resulted in a 5-fold increase, as quantitated by [alpha-32P]GTP binding, in the amount of cytosolic 24 kDa GTP-binding protein. Addition of cycloheximide plus lovastatin to cells in culture abolished the observed increase in 24 kDa GTP-binding protein. Incubation of cells with lovastatin plus [R,S]-[5-(3)H] mevalonolactone resulted in the incorporation of radioactivity into several polypeptides in both the cytosolic and particulate fractions including a polypeptide of molecular mass of 24 kDa in the cytosol. The mobility of this 24 kDa isoprenylated protein on SDS-PAGE was identical to that of the GTP-binding protein increased in response to lovastatin. However, the 24 kDa protein remained in the cytosol after undergoing isoprenylation. The 24 kDa protein was distinct from the HEL cell, G25K/CDC42Hs GTP-binding protein and the GTP-binding protein that was a substrate for botulinum toxin C3 catalyzed ADP-ribosylation. Results demonstrate that lovastatin specifically increases the expression of a 24 kDa GTP-binding protein in HEL cells and that, isoprenylation of low molecular mass GTP-binding protein(s) may have function(s) in addition to its role in the targetting of these proteins to cell membrane.

Inhibition of FcεRI-mediated Activation of Rat Basophilic Leukemia Cells by Clostridium difficile Toxin B (Monoglucosyltransferase)

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [3H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B 14C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 microg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [3H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identified in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

The Enterotoxin from Clostridium difficile (ToxA) Monoglucosylates the Rho Proteins

The enterotoxin from Clostridium difficile (ToxA) is one of the causative agents of the antibiotic-associated pseudomembranous colitis. In cultured monolayer cells ToxA exhibits cytotoxic activity to induce disassembly of the actin cytoskeleton, which is accompanied by morphological changes. ToxA-induced depolymerization of actin filaments is correlated with a decrease in the ADP-ribosylation of the low molecular mass GTP-binding Rho proteins (Just, I., Selzer, J., von Eichel-Streiber, C., and Aktories, K. (1995) J. Clin. Invest. 95, 1026-1031). Here we report on the identification of the ToxA-induced modification of Rho. Applying electrospray mass spectrometry, the mass of the modification was determined as 162 Da, which is consistent with the incorporation of a hexose into Rho. From several hexoses tested UDP-glucose selectively served as cosubstrate for ToxA-catalyzed modification. The acceptor amino acid of glucosylation was identified from a Lys-C-generated peptide by tandem mass spectrometry as Thr-37. Mutation of Thr-37 to Ala completely abolished glucosylation. The members of the Rho family (RhoA, Rac1, and Cdc42Hs) were substrates for ToxA, whereas H-Ras, Rab5, and Arf1 were not glucosylated. ToxA-catalyzed glucosylation of lysates from ToxA-pretreated rat basophilic leukemia (RBL) cells resulted in a decreased incorporation of [14C]glucose, indicating previous glucosylation in the intact cell. Glucosylation of the Rho subtype proteins appears to be the molecular mechanism by which C. difficile ToxA mediates its cytotoxic effects on cells.

Studies on the active-site structure of C3-like exoenzymes: Involvement of glutamic acid in catalysis of ADP-ribosylation

Various C3-like ADP-ribosyltransferases like Clostridium botulinum exoenzyme C3, C limosum transferase, B cereus transferase and a transferase from Staphylococcus aureus (EDIN) selectively modify the low-molecular mass GTP-binding proteins RhoA,B,C. UV-irradiation of C limosum transferase in the presence of [carbonyl- 14C]NAD resulted in radiolabeling of Glu-174. Concomitantly, ADP-ribosyltransferase and NAD glycohydrolase activities were inhibited. Site-directed mutagenesis of Glu-174 (E174D, E174Q) which resulted in more than 1000-fold reduction of enzyme activity, suggests that the glutamic acid residue is essentially involved in the catalytic action of C3-like transferases. These findings support the view that all bacterial ADP-ribosyltransferases share a similar active-site structure.

Incorporation of Rap 1b into the platelet cytoskeleton is dependent on thrombin activation and extracellular calcium.

Rap 1b is a 22-kDa low molecular mass GTP-binding protein which is both a member of the Ras superfamily and a substrate for cAMP-dependent protein kinase. Recently, evidence has been presented to show that Rap 1b is incorporated into the detergent-extracted cytoskeleton of platelets during thrombin-induced activation. The aims of this study were to compare the incorporation of Rap 1b into the detergent-extracted cytoskeleton after activation with different agonists, to examine the role of extracellular calcium on the incorporation of Rap 1b into the cytoskeleton, to investigate the relationship between the association of Rap 1b and other proteins with the cytoskeleton, and to determine the effect of phosphorylation of Rap 1b incorporation into the cytoskeleton. Platelets were activated with thrombin, A23187, phorbol myristate acetate, ADP, epinephrine, and collagen in the presence and absence of calcium. The time dependence of Rap 1b incorporation into the detergent-extracted cytoskeleton was then measured. When platelets were activated by thrombin in the presence of extracellular calcium, conditions which permit aggregation, incorporation of Rap 1b into the detergent-extracted cytoskeleton was biphasic. Approximately 20% of the total cellular Rap 1b incorporated into the cytoskeleton within seconds and was followed by a slower second phase of incorporation. In contrast, when platelets were activated by thrombin in the absence of calcium, conditions which inhibit aggregation, or by the other agents in the presence or absence of calcium, only the initial phase of Rap 1b incorporation into the cytoskeleton was measured. The incorporation of Rap 1b paralleled the incorporation of membrane glycoproteins (GP) IIb/IIIa and PECAM-1, but not the incorporation of pp60c-src. The GTPase-activating protein for Ras (Ras-GAP) did not associate with the detergent-extracted cytoskeleton. Two-dimensional isoelectric focusing SDS-polyacrylamide gel electrophoresis of the total cellular and cytoskeletal Rap 1b showed that unphosphorylated as well as phosphorylated isoforms of Rap 1b were incorporated into the cytoskeleton in the same molar ratio as was present in the intact cell. Furthermore, the rates of incorporation of phosphorylated and unphosphorylated Rap 1b into the cytoskeleton were similar. These experiments show that Rap 1b can regulate events that take place within seconds after activation, such as the initial formation of the cytoskeleton, as well as longer term changes in the cytoskeleton that occur in response to thrombin-induced aggregation. Furthermore, phosphorylation could modulate the (unknown) functions of Rap 1b as a component of the cytoskeleton.

RT-PCR cloning of Rab3 isoforms expressed in peritoneal mast cells

Rab proteins are ras-like low molecular mass GTP-binding proteins, which are postulated to act as specific regulators of membrane trafficking in exocytosis and endocytosis. We have previously shown that synthetic peptides, corresponding to the effector domain of Rab3 proteins, stimulate a complete exocytotic response in mast cells. We have used a PCR-eloning strategy to investigate the presence of mRNA encoding Rab3 in mast cells. RNA based PCR was then performed on mast cell RNA using degenerate oligonucleotide primers based on two conserved sequences among Rab3 proteins. However, no PCR products were obtained, even for proteins known to be expressed in high copy numbers in mast cells (β-actin and Fc receptor). We have found that the problem resides in the presence of mast cell secretory granule derived heparin, that copurifies with the RNA; heparin has been shown to inhibit the activity of reverse transcriptase and Taq polymerase in PCR. After treating the RNA (obtained from about 500 mast cells) with heparinase, several PCR products of varying size were obtained using primers specific for Rab3 proteins. These products were cloned and sequenced. We have found clones containing sequences that had a 100% homology at the deduced amino acid level to a portion of Rab3B and Rab3D (amino acids 16 to 83).

Post-receptor events associated with thrombin-induced platelet activation

Thrombin is by far the most potent platelet agonist. Potentially this reflects multiple intracellular processes involved in transmitting the activation signal from the initial contact with a receptor, or binding site, to the final platelet response. Platelet membranes have two putative receptors: the high affinity glycoprotein Ib, whose function remains to be clarified, and the moderate affinity autoproteolytic receptor. The autoproteolytic receptor is a member of a family of receptors, with seven transmembrane domains, which interact with GTP-binding proteins. Distal to the membrane, several forms of phospholipase C are activated and roles for both heterotrimeric and low molecular mass GTP-binding proteins have been presented. Phospholipase C acts on inositol phospholipids to generate inositol trisphosphate and diacylglycerol, both of which function as second messengers in thrombin-induced platelet activation. Inositol trisphosphate mobilizes internal calcium stores and this is accompanied, and enhanced, by an influx of calcium from the external milieu. Diacylglycerol and calcium both serve to regulate the activity of multiple protein kinases which, in turn, mediate the phosphorylated state of numerous proteins. Phosphorylation can occur on serine, threonine or tyrosine residues of target proteins and the phosphorylated state of these proteins determines the final activation of the platelet.

Post-receptor events associated with thrombin-induced platelet activation

Thrombin is by far the most potent platelet agonist. Potentially this reflects multiple intracellular processes involved in transmitting the activation signal from the initial contact with a receptor, or binding site, to the final platelet response. Platelet membranes have two putative receptors: the high affinity glycoprotein Ib, whose function remains to be clarified, and the moderate affinity autoproteolytic receptor. The autoproteolytic receptor is a member of a family of receptors, with seven transmembrane domains, which interact with GTP-binding proteins. Distal to the membrane, several forms of phospholipase C are activated and roles for both heterotrimeric and low molecular mass GTP-binding proteins have been presented. Phospholipase C acts on inositol phospholipids to generate inositol trisphosphate and diacylglycerol, both of which function as second messengers in thrombin-induced platelet activation. Inositol trisphosphate mobilizes internal calcium stores and this is accompanied, and enhanced, by an influx of calcium from the external milieu. Diacylglycerol and calcium both serve to regulate the activity of multiple protein kinases which, in turn, mediate the phosphorylated state of numerous proteins. Phosphorylation can occur on serine, threonine or tyrosine residues of target proteins and the phosphorylated state of these proteins determines the final activation of the platelet.

Low molecular mass GTP-binding proteins are secreted from mammary epithelial cells in association with lipid globules

Secretion of milk lipid globules is achieved through encapsulation of triacylglycerol-rich lipid droplets in a specialized region of apical plasma membrane of mammary epithelial cells. A class of low molecular mass GTP-binding proteins were associated tightly with the lipid globule membrane, and these proteins appeared to change from peripheral to integral membrane proteins during intracellular growth and transit of lipid globule precursors. Inclusion of GTP or GTPγS in incubation medium stimulated secretion of lipids from primary cultures of permeabilized rat mammary epithelial cells. Six polypeptides with molecular masses between 28 and 21 kDa were detected by ability to bind GTPγS following separation of lipid-globule-associated proteins by SDS-PAGE and transblotting onto nitrocellulose. That all of these polypeptides were distinct immunologically from the archetype ras was evident from lack of immunoreactivity with p 21ras G-protein monoclonal antibody in Western blots. This monoclonal antibody bound to a 23 kDa polypeptide of lipid droplets that was not detected with the GTPγS binding assay. A 25 kDa component of milk lipid globules was a potent substrate for ADP-ribosylation by botulinum toxin C3, but cholera toxin was much less effective, suggesting that this component may belong to the rac class of G-proteins. The 21 kDa component was related immunologically to ADP ribosylation factor.

Isoprenylation of Rab1B is impaired by mutations in its effector domain

Low molecular mass GTP-binding proteins encoded by the Rab gene family are posttranslationally modified by a specific geranylgeranyltransferase (GGTase II), which catalyzes the thioether linkage of geranylgeranyl isoprenoids to cysteines within one of the following carboxyl-terminal sequence motifs: GGCC, CXC, CCSN. Short peptides containing these sequences are poor substrates for isoprenylation in vitro, suggesting that structural domains remote from the carboxyl terminus are required for interactions between Rab proteins and GGTase II. To begin to define these domains, deletions and point mutations were created within the Rab1B gene, and the ability of the mutant translation products to undergo isoprenylation was evaluated in reticulocyte lysates. Deletion of amino acids 2-9 diminished but did not eliminate isoprenylation of Rab1B, suggesting that the extreme amino-terminal region is not absolutely required for interaction with GGTase II. Longer deletions in the amino-terminal region, which probably disrupt the overall conformation of Rab1B, completely prevented isoprenylation. Site-directed mutations predicted to lie in the amino-terminal variable region (Y5N), the beta 3 strand (Q60E), and Loop 7 (A110D) of the Rab1B structure did not reduce isoprenylation. However, two mutations (I41N, D44N) in the effector domain, which appears to mediate interactions with proteins that stimulate GTP hydrolysis or GDP dissociation, essentially abolished the ability of Rab1B to undergo isoprenylation. These findings imply that the effector domain plays a key role in the isoprenylation of Rab proteins, either by serving as a prenyltransferase binding site or by facilitating interactions with accessory proteins that allow Rab1B to assume a specific guanine nucleotide-dependent conformation that is recognized by GGTase II.

Identification of an ARF Type Low Molecular Mass GTP-Binding Protein in Pea (Pisum sativum)

The ARF class of low molecular mass GTP-binding proteins, originally detected as cholera toxin-activated ADP-ribosylation factors (ARF) in mammalian cells, is now known to participate in intracellular membrane vesicle trafficking. We identified a GTP-binding protein in pea plumules which resembles ARF in several respects. Like mammalian ARF, the pea protein had an apparent molecular mass of 21 kDa and was distributed mainly (∼97%) in the cytosol, with 1% and 1.5% occurring in the Golgi and microsomal fractions, respectively. In comparison, small GTP-binding proteins in the 26-30 kDa range were enriched in membranous fractions. The 21 kDa protein crossreacted strongly with an ARF class I antibody prepared against a mammalian ARF, but did not crossreact with ARF 5 (of class II) antibodies. An anti-Volvox yptl antibody did not show immunoreactivity to the 21 kDa pea protein, although it crossreacted strongly with a 28 kDa pea plumule protein. Our results strongly suggest that the 21 kDa pea protein is an analog of the ARF protein localized in the cytosol of mammalian cells. As far as we are aware, this is the first observation of an ARF protein in plants.

Isoprenoid requirement for intracellular transport and processing of murine leukemia virus envelope protein.

Lovastatin blocks the biosynthesis of the isoprenoid precursor, mevalonate. When Friend murine erythroleukemia (MEL) cells are cultured in medium containing lovastatin, the precursor of murine leukemia virus envelope glycoprotein (gPr90env) fails to undergo proteolytic processing, which normally occurs in the Golgi complex. Consequently, newly synthesized envelope proteins are not incorporated into viral particles that are shed into the culture medium. gPr90env appears to be localized in a pre-Golgi membrane compartment, based on its enrichment in subcellular fractions containing NADPH-cytochrome c reductase activity and the sensitivity of its carbohydrate chains to digestion with endoglycosidase H. Arrest of gPr90env processing occurs at concentrations of lovastatin that are not cytostatic, and the effect of the inhibitor is prevented by addition of mevalonate to the medium. The low molecular mass GTP-binding proteins, rab1p and rab6p, which are believed to function in early steps of the exocytic pathway, are normally modified posttranslationally by geranylgeranyl isoprenoids. However, in MEL cells treated with 1 microM lovastatin, nonisoprenylated forms of these proteins accumulate in the cytosol prior to arrest of gPr90env processing. These observations suggest that lovastatin may prevent viral envelope precursors from reaching the Golgi compartment by blocking the isoprenylation of rab proteins required for ER to Golgi transport.

Identification of ras and ras-related low-molecular-mass GTP-binding proteins associated with rat lung lamellar bodies.

Recent evidence from genetic experiments in yeast and from studies using guanosine triphosphate (GTP) analogues in mammalian cells suggests a key role for low-molecular-mass GTP-binding proteins (LMM-GBPs) (Mr 19 to 28 kD) in processes of intracellular vesicular sorting and secretion. Assembly and exocytosis of the lamellar body (LB), the secretory organelle of the pulmonary alveolar type 2 pneumocyte, may be regulated by LMM-GBPs. We used [alpha-32P]GTP binding to Western blotted proteins, ultraviolet crosslinking of [alpha-32P]GTP to membrane proteins, immunoblotting with specific antisera, and botulinum exoenzyme C3-catalyzed ADP ribosylation to detect LMM-GBPs in LB. With the first two techniques, we have identified six LMM-GBPs of approximately 27, 25.5, 24.5, 23, 22, and 21 kD that are enriched in a highly purified LB fraction compared with type 2 pneumocyte homogenate, crude membranes, and cytosol. Further characterization of the LB LMM-GBPs by immunoblotting revealed that ras p21 is greatly enriched in the LB fraction compared with other type 2 pneumocyte fractions. In addition, botulinum exoenzyme C3 catalyzed the ADP ribosylation of 20- to 21-kD proteins that were similarly enriched in the LB fraction. In contrast, a monospecific antibody to ADP-ribosylation factor reacted with a 19-kD protein only in the type 2 pneumocyte homogenate and cytosol fractions. Monospecific antibodies to yeast Sec4 protein and to rab 3A did not react with any type 2 pneumocyte proteins. The LMM-GBPs specifically associated with LB may participate in intracellular events required for surfactant packaging and secretion.