Three distinct F-actin binding sites in the Dictyostelium discoideum 34,000 dalton actin bundling protein

The Dictyostelium 34 kDa protein is an actin bundling protein composed of 295 amino acids. However, the region(s) of the molecule that bind actin filaments is (are) unknown. Studies of the cosedimentation of 125I-34 kDa protein and F-actin show that the 34 kDa protein binds to F-actin with positive cooperativity and Hill coefficients of 1.9 and 3.0, for filaments 4.9 microm and 0.6 microm, respectively. The Hill coefficient is larger for short filaments that are more efficiently bundled than long filaments, suggesting that one of the binding sites is used in interfilament contacts or contributes to filament orientation within the bundle. Three distinct actin binding sites were identified using a synthetic peptide, protein truncations, and a novel epitope library screening method. The ability to bind actin was assessed by 125I-F-actin overlays under denaturing and nondenaturing conditions, cosedimentation, viscometry, and pyrene-labeled actin disassembly. The three actin binding domains were identified as amino acids 1-123, 193-254, and 279-295. The 62 amino acid domain (193-254) can cosediment with F-actin. The estimated Kapp obtained by the disassembly of pyrene-labeled actin was 0.11 microM and 2.7 microM for the amino acids 1-123 and 279-295, respectively. These results identify three distinct regions of the 34 kDa protein that may contribute to the positive cooperative formation of F-actin bundles.     

Influenza virus NS1 protein interacts with the cellular 30 kDa subunit of CPSF and inhibits 3'end formation of cellular pre-mRNAs

Inhibition of the nuclear export of poly(A)-containing mRNAs caused by the influenza A virus NS1 protein requires its effector domain. Here, we demonstrate that the NS1 effector domain functionally interacts with the cellular 30 kDa subunit of CPSF, an essential component of the 3' end processing machinery of cellular pre-mRNAs. In influenza virus-infected cells, the NS1 protein is physically associated with CPSF 30 kDa. Binding of the NS1 protein to the 30 kDa protein in vitro prevents CPSF binding to the RNA substrate and inhibits 3' end cleavage and polyadenylation of host pre-mRNAs. The NS1 protein also inhibits 3' end processing in vivo, and the uncleaved pre-mRNA remains in the nucleus. Via this novel regulation of pre-mRNA 3' end processing, the NS1 protein selectively inhibits the nuclear export of cellular, and not viral, mRNAs.     

Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin

In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.     

Purification of tropomyosin from bovine adrenal medulla and its inhibitory effect on the actin severing activity of adseverin (adrenal medulla 74 kDa actin severing protein)

Tropomyosin was purified from a gelation product produced in the crude extract of bovine adrenal medulla using several column chromatographies mixed with a boiling treatment. Purified tropomyosin was a mixture of major isoforms with low molecular weights and minor isoforms of high molecular weights. Purified tropomyosin showed a dose-dependent protective effect on actin filaments against the severing activity of adseverin derived from adrenal medulla, suggesting its important role in the regulation of cell shape and structure in vivo.     

Drosophila singed, a fascin homolog, is required for actin bundle formation during oogenesis and bristle extension

Drosophila singed mutants were named for their gnarled bristle phenotype but severe alleles are also female sterile. Recently, singed protein was shown to have 35% peptide identity with echinoderm fascin. Fascin is found in actin filament bundles in microvilli of sea urchin eggs and in filopodial extensions in coelomocytes. We show that Drosophila singed is required for actin filament bundle formation in the cytoplasm of nurse cells during oogenesis; in severe mutants, the absence of cytoplasmic actin filament bundles allows nurse cell nuclei to lodge in ring canals and block nurse cell cytoplasm transport. Singed is also required for organized actin filament bundle formation in the cellular extension that forms a bristle; in severe mutants, the small disorganized actin filament bundles lack structural integrity and allow bristles to bend and branch during extension. Singed protein is also expressed in migratory cells of the developing egg chamber and in the socket cell of the developing bristle, but no defect is observed in these cells in singed mutants. Purified, bacterially expressed singed protein bundles actin filaments in vitro with the same stoichiometry reported for purified sea urchin fascin. Singed-saturated actin bundles have a molar ratio of singed/actin of approximately 1:4.3 and a transverse cross-banding pattern of 12 nm seen using electron microscopy. Our results suggest that singed protein is required for actin filament bundle formation and is a Drosophila homolog of echinoderm fascin.     

Role of incomplete dietary protein in experimental formation of gallstones

The influence exerted by the quality of alimentary protein on the composition of the secreted bile, morphology of the liver and gallbladder, as well as on the formation of gallstones was studied in experiments with rats and hamsters. It is shown that with inclusion in an experimental fat-free ration of qualitatively incomplete protein gelatin (instead of caseine) the frequency of the gallstones formation increases by almost 6 times. In animals this is attended by a reduced concentration of a number of major bile components. Upon feeding for 2 weeks on lithogenic ration with gelatin morphological investigations disclose inflammatory changes in the gallbladder wall, and after a lapse of 3 weeks--multiple exulcerations in the mucosa and a dense gross sediment in the gallbladder lumen.     

Genetic deletion of ABP-120 alters the three-dimensional organization of actin filaments in Dictyostelium pseudopods

This study extends the observations on the defects in pseudopod formation of ABP-120+ and ABP-120- cells by a detailed morphological and biochemical analysis of the actin based cytoskeleton. Both ABP-120+ and ABP-120- cells polymerize the same amount of F-actin in response to stimulation with cAMP. However, unlike ABP-120+ cells, ABP-120- cells do not incorporate actin into the Triton X-100-insoluble cytoskeleton at 30-50 s, the time when ABP-120 is incorporated into the cytoskeleton and when pseudopods are extended after cAMP stimulation in wild-type cells. By confocal and electron microscopy, pseudopods extended by ABP-120- cells are not as large or thick as those produced by ABP-120+ cells and in the electron microscope, an altered filament network is found in pseudopods of ABP-120- cells when compared to pseudopods of ABP-120+ cells. The actin filaments found in areas of pseudopods in ABP-120+ cells either before or after stimulation were long, straight, and arranged into space filling orthogonal networks. Protrusions of ABP-120- cells are less three-dimensional, denser, and filled with multiple foci of aggregated filaments consistent with collapse of the filament network due to the absence of ABP-120-mediated cross-linking activity. The different organization of actin filaments may account for the diminished size of protrusions observed in living and fixed ABP-120- cells compared to ABP-120+ cells and is consistent with the role of ABP-120 in regulating pseudopod extension through its cross-linking of actin filaments.     

Nucleotide and actin binding properties of the isolated motor domain from Dictyostelium discoideum myosin

Nucleotide and actin binding properties of the truncated myosin head (S1dC) from Dictyostelium myosin II were studied in solution using rabbit skeletal myosin subfragment 1 as a reference material. S1dC and subfragment 1 had similar affinities for ADP analogues, epsilon ADP and TNP-ADP. The complexes of epsilon ADP and BeFx or AIF4- were less stable with S1dC than with subfragment 1. Stern-Volmer constants for acrylamide quenching of S1dC complexes with epsilon ADP, epsilon ADP.AIF4- and epsilon ADP.BeFx were 2.6, 2.9 and 2.2 M-1, respectively. The corresponding values for subfragment 1 were 2.6, 1.5 and 1.1 M-1. The environment of the nucleotide binding site was probed by using a hydrophobic fluorescent probe, PPBA. PPBA was a competitive inhibitor of S1dC Ca(2+)-ATPase (Ki = 1.6 microM). The binding of nucleotides to subfragment 1 enhanced PPBA fluorescence and caused blue shifts in the wavelength of its maximum emission in the order: ATP approximately ADP.AIF4- approximately ADP.BeFx > ATP gamma S > ADP > PPi. In the case of S1dC, the effects of different nucleotides were smaller and indistinguishable from each other. S1dC bound actin tighter than S1 (Kd = 7 nM and 60 nM, respectively). The actin activated MgATPase activity of S1dC varied between preparations, and the Vmax and K(m) values ranged between 3 and 7 s-1 and 60 and 190 microM, respectively. S1dC showed lower structural stability than S1 as revealed by their thermal inactivations at 35 degrees C. These results show that the nucleotide and actin binding of S1dC and subfragment 1 are similar but there are some differences in nucleotide and phosphate analogue-induced changes and the communication between the nucleotide and actin binding sites in these proteins.     

Modulation of actin filament sliding by mutations of the SH2 cysteine in Dictyostelium myosin II

The insulin receptor gene is induced 8 to 10-fold during adipocyte differentiation. Plasmids containing the promoter, exon 1 and a portion of the first intron from either the mouse or human gene are able to modulate the expression of an insulin receptor/CAT gene 3 to 7-fold during differentiation. We have shown that several nuclear proteins from both preadipocyte and adipocyte nuclear extracts bind to two discrete sites within a 278-bp region in the 5' end of the first intron. Sequence comparison between the first intron of the human gene and the mouse gene shows two regions of sequence identity which correspond to the protein binding regions detected by DNase footprinting. One of these sites binds proteins that are enriched in adipocyte nuclear extracts and can be competed by adipose regulatory element, ARE6.     

Differential localization of alpha-actinin and the 30 kD actin-bundling protein in the cleavage furrow, phagocytic cup, and contractile vacuole of Dictyostelium discoideum

Dictyostelium discoideum amoebae possess eight different actin crosslinking proteins. Immunofluorescence microscopy has been employed in this study to investigate the intracellular localization of two of these proteins, alpha-actinin and the 30 kD actin-bundling protein, to investigate whether they are redundant, or alternatively, make distinct contributions to cell structure and movement. The 30 kD protein is concentrated in the cleavage furrow of dividing cells, while enhanced staining for alpha-actinin is not apparent in this region. By contrast, alpha-actinin is concentrated around the contractile vacuole, while the 30 kD protein is not preferentially localized in the area of this organelle. Association of alpha-actinin with the contractile vacuole was confirmed by colocalization with calmodulin, a marker of this organelle. There are temporal differences in the localization of the 30 kD protein and alpha-actinin during phagocytosis. The 30 kD protein is localized in the phagocytic cup, but disassociates from phagosomes soon after internalization [Furukawa et al., 1992: Protoplasma 169: 18-27]. alpha-actinin enters the phagocytic cup after the 30 kD protein, and remains associated with the phagosome after the 30 kD protein has disassociated. These results support the hypothesis that alpha-actinin and the 30 kD protein play distinct roles in cell structure and movement in Dictyostelium.     

Changes in actin filament organization during pseudopod formation

Fluorescent phalloidin has been introduced into Dicytostelium amoebae in order to visualize dynamic changes in the localization of F-actin during pseudopod extension. Phalloidin was initially localized to the peripheral cortex of the cell. Newly formed pseudopods were not fluorescent, indicating that phalloidin was tightly bound to existing F-actin filaments and could not rapidly relocalize to newly formed filaments. As pseudopod extension proceeded, the fluorescent signal disappeared from the region directly underlying the expansion zone, leaving a gap in the actin cortex. Similar results were obtained in both wild-type cells and those lacking myosin II heavy chain. The disappearance of the fluorescent signal from the cortical region underlying the new pseudopod is presumed to be due to breakdown of the actin cortex and dispersion of the remnants. These results suggest that new pseudopods are not built upon the existing actin cortex but rather that the cortex is locally solated as part of the construction of the new actin network.     

200 kDa and 160 kDa neurofilament protein phosphatase resistance following in vivo aluminum chloride exposure

We have used time-course dephosphorylation experiments and two dimensional isoelectric focusing to assess the phosphorylation state of neurofilament (NF) proteins following the intracisternal inoculation of AlCl3. Littermates of New Zealand white rabbits, age 5-6 weeks, were inoculated with either 1000, 750, 500, 250 or 100 micrograms AlCl3 in 0.9% NaCl or 0.9% NaCl alone, killed 48 hours later and the NF-enriched cytoskeletal fraction isolated from the spinal cord. Neurofilamentous inclusions did not occur following inoculums of 100 or 250 micrograms AlCl3, but thereafter developed in increasing quantities in a dosage-dependent manner. Incubation of the NF-enriched fraction with E. Coli. alkaline phosphatase (enzyme: substrate 1:50) induced a replacement of the highly phosphorylated 200 kDa isoform of NFH with a more poorly phosphorylated 170 kDa isoform, confirmed by immunoblot analysis. This reaction was complete within 20 minutes with NF derived from NaCl, 100 or 250 micrograms AlCl3 inoculated rabbits and within 30 minutes for 500 micrograms AlCl3 inoculums. However, residual highly phosphorylated NFH isoforms persisted at 60 minutes for 750 micrograms inoculums and 90 minutes for that derived from 1000 micrograms AlCl3 inoculums. A similar inhibition of phosphatase activity was observed for NFM. Following two dimensional electrophoresis of the NF-enriched isolate, no alteration in the net phosphorylation state of individual NF subunit proteins was observed--regardless of the inoculum. These results demonstrate a dose-dependent induction of neurofilamentous inclusions in spinal motor neurons following intracisternal AlCl3 inoculation accompanied by increasing phosphatase resistance without a demonstrable alteration in NF net phosphorylation state.     

Role of cotranslational disulfide bond formation in the folding of the hemagglutinin-neuraminidase protein of Newcastle disease virus

The role of cotranslational disulfide bond formation in the folding pathway of the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus was explored. Electrophoresis of pulse-labeled HN protein in the presence or absence of reducing agent showed that, characteristic of many glycoproteins, the nascent HN protein contains intramolecular disulfide bonds. As reported by Braakman et al. (EMBO J. 11, 1717-1722, 1992), incubation of cells in dithiothreitol (DTT) blocked the formation of these bonds. Removal of DTT after a pulse-label allowed for the subsequent formation of intramolecular disulfide bonds and folding of the molecule as assayed by the appearance of conformationally sensitive antigenic sites and by the formation of disulfide-linked dimers. However, the t1/2 for the formation of a conformationally sensitive antigenic site after synthesis in the presence of DTT was over twice that of the control. Furthermore, the order of appearance of the antigenic sites was different from the control, suggesting that inhibition of cotranslational disulfide bond formation altered the folding pathway of the protein. Similar results were obtained in a cell-free system containing membranes. The HN protein forced to form intramolecular disulfide bonds posttranslationally had no detectable neuraminidase or cell attachment activity, suggesting that the protein had an abnormal conformation.     

Regulation of the cortical actin cytoskeleton in budding yeast by twinfilin, a ubiquitous actin monomer-sequestering protein

Here we describe the identification of a novel 37-kD actin monomer binding protein in budding yeast. This protein, which we named twinfilin, is composed of two cofilin-like regions. In our sequence database searches we also identified human, mouse, and Caenorhabditis elegans homologues of yeast twinfilin, suggesting that twinfilins form an evolutionarily conserved family of actin-binding proteins. Purified recombinant twinfilin prevents actin filament assembly by forming a 1:1 complex with actin monomers, and inhibits the nucleotide exchange reaction of actin monomers. Despite the sequence homology with the actin filament depolymerizing cofilin/actin-depolymerizing factor (ADF) proteins, our data suggests that twinfilin does not induce actin filament depolymerization. In yeast cells, a green fluorescent protein (GFP)-twinfilin fusion protein localizes primarily to cytoplasm, but also to cortical actin patches. Overexpression of the twinfilin gene (TWF1) results in depolarization of the cortical actin patches. A twf1 null mutation appears to result in increased assembly of cortical actin structures and is synthetically lethal with the yeast cofilin mutant cof1-22, shown previously to cause pronounced reduction in turnover of cortical actin filaments. Taken together, these results demonstrate that twinfilin is a novel, highly conserved actin monomer-sequestering protein involved in regulation of the cortical actin cytoskeleton.     

The protease-protected 30 kDa domain of SecA is largely inaccessible to the membrane lipid phase

SecA binds to the inner membrane of Escherichia coli through low affinity lipid interactions or with high affinity at SecYEG, the integral domain of preprotein translocase. Upon addition of preprotein and nucleotide, a 30 kDa domain of SecYEG-bound SecA is protected from proteolysis via membrane insertion. Such protection could result from some combination of insertion into the lipid phase, into a proteinaceous environment or across the membrane. To assess the exposure of SecYEG-bound SecA to membrane lipids, a radiolabeled, photoactivatable and lipid-partitioning crosslinker, 3-trifluoromethyl-3-(m[125I]iodophenyl) diazirine benzoic acid ester, was incorporated into inner membrane vesicles. The 30 kDa domain of SecYEG-bound SecA, inserted into the membrane in response to translocation ligands, is 18-fold less labeled than SecY, which is labeled effectively. In contrast, incorporation of the purified 30 kDa SecA fragment into crosslinker-containing detergent micelles or addition of detergent to crosslinker-containing membranes bearing the protease-protected SecA domain readily allows for labeling of this domain. We propose that the protease-inaccessible 30 kDa SecA domain is shielded from the fatty acyl membrane phase by membrane-spanning SecYEG helices and/or is largely exposed to the periplasm.     

Role of the actin cytoskeleton on epithelial Na+ channel regulation

The regulatory role of actin filament organization on epithelial Na+ channel activity is reviewed in this report. The actin cytoskeleton, consisting of actin filaments and associated actin-binding proteins, is essential to various cellular events including the maintenance of cell shape, the onset of cell motility, and the distribution and stability of integral membrane proteins. Functional interactions between the actin cytoskeleton and specific membrane transport proteins are, however, not as well understood. Recent studies from our laboratory have determined that dynamic changes in the actin cytoskeletal organization may represent a novel signaling mechanism in the regulation of ion transport in epithelia. This report summarizes work conducted in our laboratory leading to an understanding of the molecular steps associated with the regulatory role of the actin-based cytoskeleton on epithelial Na+ channel function. The basis of this interaction lies on the regulation by actin-binding proteins and adjacent structures, of actin filament organization which in turn, modulates ion channel activity. The scope of this interaction may extend to such relevant cellular events as the vasopressin response in the kidney.     

Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator

The anion-selective channel CFTR (cystic fibrosis transmembrane conductance regulator), whose dysfunction is responsible for the onset of cystic fibrosis, is regulated by cAMP through the activation of protein kinase A (PKA). The nature of this activation process is unknown. In the present study, patch-clamp techniques were applied to both mouse mammary adenocarcinoma cells expressing human epithelial CFTR (CFTR cells) and cultured neonatal rat ventricular myocytes (NRVM), to determine whether CFTR is modulated by the actin cytoskeleton, and whether the actin cytoskeleton may be implicated in the cAMP-stimulated activation of the channel protein. Acute changes in the actin cytoskeleton by addition of cytochalasin D (CD) activated whole-cell currents in CFTR cells and NRVM. Addition of actin to excised, inside-out patches also activated CFTR. A functional characterization of CFTR in either cell type included cAMP-induced, linear whole-cell and single-channel currents in symmetrical Cl-, permeability to ATP, and inhibition by either diphenylamine-carboxylate (DPC) or a monoclonal antibody raised against CFTR. Incubation of CFTR cells and NRVM with CD for over 6 h prevented CFTR activation either by the cAMP pathway under whole-cell conditions or by PKA under excised inside-out conditions. Thus a complete derangement of the actin cytoskeleton prevents the cAMP-dependent activation of CFTR. CFTR activation, however, was restored by subsequent addition of actin. In summary, changes in actin filament organization modulate CFTR channel activity by a mechanism entailing a direct interaction between actin filaments and CFTR.     

Immunoblot studies in the differential diagnosis of porcine brucellosis: an immunodominant 62 kDa protein is related to the mycobacterial 65 kDa heat shock protein (HSP-65)

Smooth Brucella spp. share certain lipopolysaccharide antigens with other bacteria, resulting in serological cross-reactions which can prevent the definitive diagnosis of brucellosis. To identify other antigens with serodiagnostic potential, immunoblot studies following sodium dodecyl sulphate-polyacrylamide gel electrophoresis were carried out. Sera from pigs experimentally infected with Brucella suis and naturally infected feral pigs, sera from pigs from a farm with a known history of Yersinia enterocolitica 0:9 infection, Brucella Complement Fixation Test (CFT) reactor pigs (aetiology unknown) and pigs from consistently Brucella CFT negative farms were examined. Although B. suis infected pigs recognized a total of nine B. melitensis antigens, individual pigs rarely recognized more than three antigens in the range. A 62 kDa antigen was recognized by the majority (73%) of the Brucella infected pigs, but only by 10 to 23% of pigs from the other groups. This antigen was shown to be the Brucella homologue of the ubiquitous 65 kDa heat shock protein (HSP-65) family by immunoblot studies with 14 monoclonal antibodies to the Mycobacterium leprae HSP-65. Only four of these monoclones (Y1.2, ML-30, D7C and IIIC8) identified the B. melitensis 62 kDa protein suggesting that unshared, potentially Brucella specific, regions exist. Sera from Y. enterocolitica 0:9 infected pigs, CFT reactor pigs (aetiology unknown), CFT negative pigs and hyperimmune pig serum raised to Y. enterocolitica 0:9 also recognized B. melitensis antigens, most notably a 17 kDa protein. This antigen appears to be a common cross-reactive protein.     

Activation of the mitogen-activated protein kinase ERK2 by the chemoattractant folic acid in Dictyostelium

The Dictyostelium MAP kinase ERK2 is activated by extracellular cAMP in aggregation-competent cells and is required for receptor activation of adenylyl cyclase (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P. N., and Firtel, R. A. (1996) J. Biol. Chem. 271, 3351-3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W. (1995) J. Cell Biol. 128, 405-413). This cAMP-dependent activation of ERK2 is mediated by the serpentine, G protein-coupled cAMP receptors. However, ERK2 activation by cAMP is at least partially heterotrimeric G protein-independent, with a level of activation in cells lacking the sole Gbeta subunit or the G protein-coupled cAMP receptors-coupled Galpha2 subunit that is approximately 50% that of wild-type cells (Maeda, M., Aubry, L., Insall, R., Gaskins, C., Devreotes, P. N., and Firtel, R. A. (1996) J. Biol. Chem. 271, 3351-3354; Segall, J., Kuspa, A., Shaulsky, G., Ecke, M., Maeda, M., Gaskins, C., Firtel, R., and Loomis, W. (1995) J. Cell Biol. 128, 405-413). Folic acid, a chemoattractant in the vegetative cells that enables amoebae to find bacteria in the wild, also triggers the activation of adenylyl cyclase, which is impaired in the vegetative cells lacking the Galpha protein subunit Galpha4 (Hadwiger, J., Lee, S., and Firtel, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10566-10570). In this study, we show that folic acid activates ERK2 in developmentally regulated manner and is required for ERK2 stimulation of adenylyl cyclase activity. Maximum levels of folate-stimulated ERK2 activity occur in cells from very early in development, prior to aggregation, and again at the tipped aggregate stages, corresponding to the stages in which folate receptors and the coupled Galpha subunit Galpha4 are maximally expressed. During the activation by folic acid, ERK2 is phosphorylated on tyrosine residue(s) and contemporaneously shows a mobility shift on SDS-PAGE. Interestingly, this activation is not elicited in the absence of Gbeta subunits, in contrast to the response to cAMP. This response also requires the Galpha4 subunit known to be required for other folic acid-mediated responses (Hadwiger, J., Lee, S., and Firtel, R. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10566-10570). Furthermore, we show that the activation of ERK2 by cAMP is independent of the Galpha4 subunit, while the activation of ERK2 by folate is independent of Galpha2. Taken together, these data indicate that there are at least two pathways of ERK2 activation, heterotrimeric G protein-dependent and -independent pathways.     

A novel multi-functional chloroplast protein: identification of a 40 kDa immunophilin-like protein located in the thylakoid lumen

We describe the identification of the first immunophilin associated with the photosynthetic membrane of chloroplasts. This complex 40 kDa immunophilin, designated TLP40 (thylakoid lumen PPIase), located in the lumen of the thylakoids, was found to play a dual role in photosynthesis involving both biogenesis and intraorganelle signalling. It originates in a single-copy nuclear gene, is made as a precursor of 49.2 kDa with a bipartite lumenal targeting transit peptide, and is characterized by a structure including a cyclophilin-like C-terminal segment of 20 kDa, a predicted N-terminal leucine zipper and a potential phosphatase-binding domain. It can exist in different oligomeric conformations and attach to the inner membrane surface. It is confined predominantly to the non-appressed thylakoid regions, the site of protein integration into the photosynthetic membrane. The isolated protein possesses peptidyl-prolyl cis-trans isomerase protein folding activity characteristic of immunophilins, but is not inhibited by cyclosporin A. TLP40 also exerts an effect on dephosphorylation of several key proteins of photosystem II, probably as a constituent of a transmembrane signal transduction chain. This first evidence for a direct role of immunophilins in a photoautotrophic process suggests that light-mediated protein phosphorylation in photosynthetic membranes and the role of the thylakoid lumen are substantially more complex than anticipated.