Showing papers by "Kettering University published in 1992"

Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding

Abstract: The main stress proteins of Escherichia coli function in an ordered protein-folding reaction. DnaK (heat-shock protein 70) recognizes the folding polypeptide as an extended chain and cooperates with DnaJ in stabilizing an intermediate conformational state lacking ordered tertiary structure. Dependent on GrpE and ATP hydrolysis, the protein is then transferred to GroEL (heat-shock protein 60) which acts catalytically in the production of the native state. This sequential mechanism of chaperone action may represent an important pathway for the folding of newly synthesized polypeptides.

Molecular dissection of the secretory pathway

Abstract: A combination of biochemistry in animal cell-free systems and genetics in yeast is revealing the molecular machinery of the secretory pathway of eukaryotes. Transporting vesicles have a simple coat structure and employ a general mechanism for fusion that is conserved in evolution.

Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF

Abstract: A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.

Chaperonin-mediated protein folding: GroES binds to one end of the GroEL cylinder, which accommodates the protein substrate within its central cavity.

Abstract: The mechanism of GroEL (chaperonin)-mediated protein folding is only partially understood. We have analysed structural and functional properties of the interaction between GroEL and the co-chaperonin GroES. The stoichiometry of the GroEL 14mer and the GroES 7mer in the functional holo-chaperonin is 1:1. GroES protects half of the GroEL subunits from proteolytic truncation of the approximately 50 C-terminal residues. Removal of this region results in an inhibition of the GroEL ATPase, mimicking the effect of GroES on full-length GroEL. Image analysis of electron micrographs revealed that GroES binding triggers conspicuous conformational changes both in the GroES adjacent end and at the opposite end of the GroEL cylinder. This apparently prohibits the association of a second GroES oligomer. Addition of denatured polypeptide leads to the appearance of irregularly shaped, stain-excluding masses within the GroEL double-ring, which are larger with bound alcohol oxidase (75 kDa) than with rhodanese (35 kDa). We conclude that the functional complex of GroEL and GroES is characterized by asymmetrical binding of GroES to one end of the GroEL cylinder and suggest that binding of the substrate protein occurs within the central cavity of GroEL.

Function in protein folding of TRiC, a cytosolic ring complex containing TCP-1 and structurally related subunits.

Abstract: T-complex polypeptide 1 (TCP-1) was analyzed as a potential chaperonin (GroEL/Hsp60) equivalent of the eukaryotic cytosol. We found TCP-1 to be part of a hetero-oligomeric 970 kDa complex containing several structurally related subunits of 52-65 kDa. These members of a new protein family are assembled into a TCP-1 ring complex (TRiC) which resembles the GroEL double ring. The main function of TRiC appears to be in chaperoning monomeric protein folding: TRiC binds unfolded polypeptides, thereby preventing their aggregation, and mediates the ATP-dependent renaturation of unfolded firefly luciferase and tubulin. At least in vitro, TRiC appears to function independently of a small co-chaperonin protein such as GroES. Folding of luciferase is mediated by TRiC but not by GroEL/ES. This suggests that the range of substrate proteins interacting productively with TRiC may differ from that of GroEL. We propose that TRiC mediates the folding of cytosolic proteins by a mechanism distinct from that of the chaperonins in specific aspects.

Differential expression and processing of two cell associated forms of the kit-ligand: KL-1 and KL-2.

Abstract: The c-kit ligand, KL, and its receptor, the proto-oncogene c-kit are encoded, respectively, at the steel (Sl) and white spotting (W) loci of the mouse. Both Sl and W mutations affect cellular targe...

Anatomy of the steroid receptor zinc finger region

Abstract: I. Introduction LIKE many transcriptional regulatory proteins, nuclear hormone receptors are single polypeptides that are organized into relatively discrete functional domains (1). This common domain organization groups these receptors into a superfamily of functionally and most likely structurally related, hormonally regulable, transcription factors. The superfamily includes receptors for steroid hormones, such as glucocorticoids, progesterone, estrogen, aldosterone, and androgens, as well as hormonal forms of vitamins A and D, thyroid hormone, and peroxisomal activators (2–6). Several other receptorlike proteins whose ligands have yet to be identified have recently been isolated utilizing molecular cloning techniques. In the same way, several putative nuclear receptors from Drosophila that appear to act as regulators of early development (Refs. 7–10; reviewed in Ref. 11) have been identified, including a receptor for the insect steroid ecdysone (12). Upon association with a particular ligand, nuclear re...

Protein Folding in the Cell: The Role of Molecular Chaperones Hsp70 and Hsp60

Abstract: THE MEMBERS OFTHE HSP70 FAMILY •• 298 Unfolding Is Required for Membrane Translocation of Proteins 299 Cytoso/ic Hsp70 Stabilizes Precursor Proteins for Translocation 300 Organellar Hsp70 in Protein Translocation and Folding 301 Requirement of Hsp70 for Protein Assembly in the ER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302 Molecular Mechanism of Hsp70 Action .... .. . . . . . . . . . .. . .. . . .. ... . . . ........ 303 THE MEMBERS OF THE HSP60 FAMILY ... ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 306 Multiple Functions of Mitochondrial Hsp60 308 Molecular Mechanism of Chaperon in Action . .. . . . 311

Prevention of protein denaturation under heat stress by the chaperonin Hsp60

Abstract: The increased synthesis of heat shock proteins is a ubiquitous physiological response of cells to environmental stress. How these proteins function in protecting cellular structures is not yet understood. The mitochondrial heat shock protein 60 (Hsp60) has now been shown to form complexes with a variety of polypeptides in organelles exposed to heat stress. The Hsp60 was required to prevent the thermal inactivation in vivo of native dihydrofolate reductase (DHFR) imported into mitochondria. In vitro, Hsp60 bound to DHFR in the course of thermal denaturation, preventing its aggregation, and mediated its adenosine triphosphate-dependent refolding at increased temperatures. These results suggest a general mechanism by which heat shock proteins of the Hsp60 family stabilize preexisting proteins under stress conditions.

A multisubunit particle implicated in membrane fusion.

Abstract: The N-ethylmaleimide sensitive fusion protein (NSF) is required for fusion of lipid bilayers at many locations within eukaryotic cells. Binding of NSF to Golgi membranes is known to require an integral membrane receptor and one or more members of a family of related soluble NSF attachment proteins (alpha-, beta-, and gamma-SNAPs). Here we demonstrate the direct interaction of NSF, SNAPs and an integral membrane component in a detergent solubilized system. We show that NSF only binds to SNAPs in the presence of the integral receptor, resulting in the formation of a multisubunit protein complex with a sedimentation coefficient of 20S. Particle assembly reveals striking differences between members of the SNAP protein family; gamma-SNAP associates with the complex via a binding site distinct from that used by alpha- and beta-SNAPs, which are themselves equivalent, alternative subunits of the particle. Once formed, the 20S particle is subsequently able to disassemble in a process coupled to the hydrolysis of ATP. We suggest how cycles of complex assembly and disassembly could help confer specificity to the generalized NSF-dependent fusion apparatus.

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack.

Abstract: We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.

Mapping of the protein import machinery in the mitochondrial outer membrane by crosslinking of translocation intermediates.

Abstract: MITOCHONDRIA contain a complex machinery for the import of nuclear-encoded proteins1,2. Receptor proteins exposed on the outer membrane surface are required for the specific binding of precursor proteins to mitochondria, either by binding of cytosolic signal recognition factors or by direct recognition of the precursor polypeptides1–5. Subsequently, the precursors are inserted into the outer membrane at the general insertion site GIP (general insertion protein)6–10. Here we report the analysis of receptors and GIP by crosslinking of translocation intermediates and by coimmunoprecipitation. Surface-accumulated precursors were cross-linked to the receptors MOM19 and MOM72, suggesting a direct interaction of preproteins with surface receptors. We identified three novel mitochondrial outer membrane proteins, MOM7, MOMS, and MOM30 that, together with the previously identified MOM38, seem to form the GIP site and are present in the mitochondrial receptor complex.

Signal transduction by Fc gamma RIII (CD16) is mediated through the gamma chain.

Abstract: To determine the functional role of the two isoforms of Fc gamma RIII (CD16) (IIIA, IIIB), the signal transduction capabilities of wild-type and mutant forms of these receptors were analyzed in transfected lymphoid, myeloid, and fibroblastic cell lines. Functional reconstitution of receptor signalling was observed in hematopoietic T and mast cells, and was absent in nonhematopoietic (CHO) cells. Fc gamma RIIIA, a hetero-oligomeric receptor composed of a ligand-binding subunit alpha and dimeric gamma chains, generated both proximal and distal responses in Jurkat and P815 cells, typical of what is seen in natural killer cells and macrophages upon receptor activation. In contrast, Fc gamma RIIIB, which is normally attached to the cell surface via a glycosyl-phosphatidylinositol anchor, was incapable of transducing signals. After crosslinking, Fc gamma RIIIA signalling was dependent only upon the gamma chain. Fc gamma RIIIA chimeras in which the alpha subunit transmembrane and cytoplasmic domains were substituted with the corresponding gamma chain sequences functioned as well as wild-type hetero-oligomeric receptors. These data indicate that the ability of the Fc gamma RIIIA complex to activate the appropriate pathways for cell activation is cell-type restricted and independent of the transmembrane and cytoplasmic domains of the alpha subunit. The presence of the gamma chain is responsible for the assembly of, as well as the signal transduction by, the functional cell surface complex.

Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells.

Abstract: Overexpression of sequences corresponding to the major Rev-binding site in the Rev response element of human immunodeficiency virus type 1 (HIV-1) (RRE decoys) was used to render cells resistant to HIV-1 replication. This was accomplished by the use of a chimeric tRNA-RRE transcription unit in a double-copy murine retroviral vector to express high levels of HIV-1 RRE-containing transcripts in CEM SS cells. Replication of HIV-1 was inhibited more than 90% in cells expressing chimeric tRNA-RRE transcripts, as determined by in situ immunofluorescence analysis and a p24 antigen ELISA test. Analysis of RNA from HIV-1-infected cells suggests that expression of RRE-containing sequences in CEM SS cells inhibits HIV-1 replication by interfering with Rev function, presumably by competing for Rev binding to its physiological target. The use of a subfragment of RRE as decoy RNA reduces the likelihood that essential cellular factors will be sequestered in cells expressing the decoy RNA. Thus, use of RRE-based decoy RNA to inhibit HIV-1 replication may represent a safer alternative to the use of TAR decoy RNA.

Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases.

Abstract: Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer of this protein. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene [Fathi, Z. & Condit, R.C. (1991) Virology 181, 258-272] define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery.

Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates.

Abstract: We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs). Three erythrocytic stage transcription units are clustered in a 40 kb region. Two of these genes are closely linked, separated by less than 2 kb. Nuclear run-on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic. Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates. These studies represent the first description of the minimal and necessary cis-acting elements for transcription termination and initiation in this protozoan parasite.

A freeze-frame view of eukaryotic transcription during elongation and capping of nascent mRNA.

Abstract: Ribonuclease footprinting of nascent messenger RNA within ternary complexes of vaccinia RNA polymerase revealed an RNA binding site that encompasses an 18-nucleotide RNA segment. The dimensions of the binding site did not change as the polymerase moved along the template. Capping of the 59 end of the RNA was cotranscriptional and was confined to nascent chains 31 nucleotides or greater in length. Purified capping enzyme formed a binary complex with RNA polymerase in solution in the absence of nucleic acid. These findings suggest a mechanism for cotranscriptional establishment of messenger RNA identity in eukaryotes.

c-kit receptor and ligand expression in postnatal development of the mouse cerebellum suggests a function for c-kit in inhibitory interneurons.

Abstract: The c-kit receptor and its cognate ligand, KL, are encoded at the white spotting locus (W) and the steel locus (Sl) of the mouse, respectively. Sl and W mutations affect the same cellular targets in melanogenesis, gametogenesis and hematopoiesis during embryonic development and in adult life. c-kit is expressed in cellular targets of W and Sl mutations, whereas KL is expressed in the microenvironment of these targets. c-kit and KL, however, are also expressed in tissues and cell types that are not targets of W and Sl mutations, including the brain. The cerebellum contains a small number of neural cell types whose developmental origins, pathways of migration, and synaptic contacts are known. We have investigated the patterns of expression of the c-kit and KL RNA and protein products in postnatal cerebellar development of the mouse. In the adult cerebellum, c-kit RNA and protein expression was evident in basket, stellate, and Golgi neurons. Most strikingly, the c- kit protein is expressed in the basket cell axons that form “basket” and “pinceau” structures entwining the Purkinje cell soma and the initial segment of the Purkinje cell axon. KL RNA expression was found in Purkinje cells, and the KL protein was detected in Purkinje cell bodies and dendrites. Soluble KL protein was also present in c-kit- expressing basket, stellate, and Golgi cells, presumably as a result of internalization of ligand-receptor complexes. During postnatal development, c-kit and KL RNA and protein expression in Golgi and Purkinje neurons, respectively, was evident by day 0 and persisted subsequently. c-kit expression in basket and stellate cells was detected from their time of birth, starting at day 4. These results suggest a role for the c-kit receptor system in postnatal development of the cerebellum.

The beta subunit of the Fc epsilon RI is associated with the Fc gamma RIII on mast cells.

Abstract: Fc epsilon RI is a tetrameric receptor, composed of a ligand recognition subunit, alpha, a beta chain, and dimeric gamma chains. Previous studies have indicated that the dimeric gamma chain is associated with Fc gamma RIIIA (CD16) on natural killer cells and macrophages as well as the clonotypic T cell receptor. Here we show that in mast cells, in addition to the dimeric gamma chains, the beta subunit is associated not only with Fc epsilon RI, but also with Fc gamma RIIIA. Functional reconstitution studies with a mastocytoma cell line indicate that Fc gamma RIIIA composed of alpha, beta, and gamma subunits has the capacity for signal transduction. These studies suggest that through the association of alternative ligand recognition subunits (alpha epsilon, alpha gamma), a common signal transduction complex (beta gamma 2) mediates similar biochemical and effector functions in response to immunoglobulin G (IgG) and IgE.

Studies on the initiation of simian virus 40 replication in vitro: RNA primer synthesis and its elongation.

Abstract: DNA primase-dependent synthesis of oligoribonucleotides 10-15 nucleotides long was observed in the presence of ATP, UTP, GTP, and CTP by using the purified components of the simian virus 40 (SV40) DNA replication system. The DNA primase-catalyzed reaction required the SV40 large tumor antigen (T antigen), DNA polymerase alpha (pol-alpha), the three-subunit human single-stranded DNA binding protein (HSSB), and topoisomerase I. The synthesis of small RNAs was unaffected by the addition of activator 1, proliferating cell nuclear antigen, and DNA polymerase delta, proteins that can support extensive leading-strand synthesis. The RNA primers were derived predominantly from transcription of the lagging-strand template, even after prolonged incubation, indicating that the leading strand did not serve as a template. When the four dNTPs were added after oligoribonucleotide synthesis, pol-alpha extended the RNA primers hybridized to SV40 DNA. Pulse-chase experiments revealed that the small RNA chains were elongated to Okazaki-sized products. T7 DNA polymerase was also shown to rapidly extend oligoribonucleotide primers in the presence of aphidicolin or antibodies against pol-alpha, conditions under which pol-alpha was markedly inhibited. These findings suggest that interactions between T antigen, pol-alpha-primase, and HSSB position the pol-alpha-primase complex on the lagging-strand template for RNA primer synthesis.

Genetic complexity of regulatory mutants defective for HLA class II gene expression.

Abstract: MHC (called HLA in man) class II genes play an essential role in cell-mediated immunity. Absence of HLA class II Ag on B lymphocytes is the basis of some congenital immunodeficiencies (CID). We have studied CID by generating transient heterokaryons from cell lines of such patients, and we report that the mutations fall into four complementation groups. In addition, fusions with the HLA class II deletion mutant 721.180 indicate that the genetic defects for each group in HLA class II expression map outside the HLA class II region. A small HLA-DRA promoter fragment is sufficient to drive expression of a reporter gene in normal B cell lines, but expression from the same construct is clearly reduced in mutant cell lines representative of all four complementation groups. This confirms earlier results that indicate defective transcription of HLA class II genes in the class II- CID mutant cell lines. Analysis of proteins that bind to the DRA promoter in nuclear extracts of the mutants suggests that complexes recognizing distinct elements of the DRA promoter may be quantitatively decreased in different mutants. In addition, we show that nuclear extracts from two groups fail to transcribe a DRA promoter construct in vitro accurately reflecting their DRA- phenotypes. In contrast, nuclear extracts from another mutant, RJ2.2.5, transcribe the DRA construct, albeit at a reduced level. Finally, though cell lines from different groups complement each other in vivo, no complementation was observed by mixing extracts for transcription in vitro.