ERp57 Functions as a Subunit of Specific Complexes Formed with the ER Lectins Calreticulin and Calnexin

ERp57 is a lumenal protein of the endoplasmic reticulum (ER) and a member of the protein disulfide isomerase (PDI) family. In contrast to archetypal PDI, ERp57 interacts specifically with newly synthesized glycoproteins. In this study we demonstrate that ERp57 forms discrete complexes with the ER lectins, calnexin and calreticulin. Specific ERp57/calreticulin complexes exist in canine pancreatic microsomes, as demonstrated by SDS-PAGE after cross-linking, and by native electrophoresis in the absence of cross-linking. After in vitro translation and import into microsomes, radiolabeled ERp57 can be cross-linked to endogenous calreticulin and calnexin while radiolabeled PDI cannot. Likewise, radiolabeled calreticulin is cross-linked to endogenous ERp57 but not PDI. Similar results were obtained in Lec23 cells, which lack the glucosidase I necessary to produce glycoprotein substrates capable of binding to calnexin and calreticulin. This observation indicates that ERp57 interacts with both of the ER lectins in the absence of their glycoprotein substrate. This result was confirmed by a specific interaction between in vitro synthesized calreticulin and ERp57 prepared in solution in the absence of other ER components. We conclude that ERp57 forms complexes with both calnexin and calreticulin and propose that it is these complexes that can specifically modulate glycoprotein folding within the ER lumen.

Regulation of G2/M Progression by the STE Mitogen-activated Protein Kinase Pathway in Budding Yeast Filamentous Growth

Inoculation of diploid budding yeast onto nitrogen-poor agar media stimulates a MAPK pathway to promote filamentous growth. Characteristics of filamentous cells include a specific pattern of gene expression, elongated cell shape, polar budding pattern, persistent attachment to the mother cell, and a distinct cell cycle characterized by cell size control at G2/M. Although a requirement for MAPK signaling in filamentous gene expression is well established, the role of this pathway in the regulation of morphogenesis and the cell cycle remains obscure. We find that ectopic activation of the MAPK signal pathway induces a cell cycle shift to G2/M coordinately with other changes characteristic of filamentous growth. These effects are abrogated by overexpression of the yeast mitotic cyclins Clb1 and Clb2. In turn, yeast deficient for Clb2 or carrying cdc28-1N, an allele of CDK defective for mitotic functions, display enhanced filamentous differentiation and supersensitivity to the MAPK signal. Importantly, activation of Swe1-mediated inhibitory phosphorylation of Thr-18 and/or Tyr-19 of Cdc28 is not required for the MAPK pathway to affect the G2/M delay. Mutants expressing a nonphosphorylatable mutant Cdc28 or deficient for Swe1 exhibit low-nitrogen-dependent filamentous growth and are further induced by an ectopic MAPK signal. We infer that the MAPK pathway promotes filamentous growth by a novel mechanism that inhibits mitotic cyclin/CDK complexes and thereby modulates cell shape, budding pattern, and cell-cell connections.

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.

Induction of a G2-Phase Arrest in Xenopus Egg Extracts by Activation of p42 Mitogen-activated Protein Kinase

Previous work has established that activation of Mos, Mek, and p42 mitogen-activated protein (MAP) kinase can trigger release from G2-phase arrest in Xenopus oocytes and oocyte extracts and can cause Xenopus embryos and extracts to arrest in mitosis. Herein we have found that activation of the MAP kinase cascade can also bring about an interphase arrest in cycling extracts. Activation of the cascade early in the cycle was found to bring about the interphase arrest, which was characterized by an intact nuclear envelope, partially condensed chromatin, and interphase levels of H1 kinase activity, whereas activation of the cascade just before mitosis brought about the mitotic arrest, with a dissolved nuclear envelope, condensed chromatin, and high levels of H1 kinase activity. Early MAP kinase activation did not interfere significantly with DNA replication, cyclin synthesis, or association of cyclins with Cdc2, but it did prevent hyperphosphorylation of Cdc25 and Wee1 and activation of Cdc2/cyclin complexes. Thus, the extracts were arrested in a G2-like state, unable to activate Cdc2/cyclin complexes. The MAP kinase-induced G2 arrest appeared not to be related to the DNA replication checkpoint and not to be mediated through inhibition of Cdk2/cyclin E; evidently a novel mechanism underlies this arrest. Finally, we found that by delaying the inactivation of MAP kinase during release of a cytostatic factor-arrested extract from its arrest state, we could delay the subsequent entry into mitosis. This finding suggests that it is the persistence of activated MAP kinase after fertilization that allows the occurrence of a G2-phase during the first mitotic cell cycle.

Interactions of RecF protein with RecO, RecR, and single-stranded DNA binding proteins reveal roles for the RecF–RecO–RecR complex in DNA repair and recombination

The products of the recF, recO, and recR genes are thought to interact and assist RecA in the utilization of single-stranded DNA precomplexed with single-stranded DNA binding protein (Ssb) during synapsis. Using immunoprecipitation, size-exclusion chromatography, and Ssb protein affinity chromatography in the absence of any nucleotide cofactors, we have obtained the following results: (i) RecF interacts with RecO, (ii) RecF interacts with RecR in the presence of RecO to form a complex consisting of RecF, RecO, and RecR (RecF–RecO–RecR); (iii) RecF interacts with Ssb protein in the presence of RecO. These data suggested that RecO mediates the interactions of RecF protein with RecR and with Ssb proteins. Incubation of RecF, RecO, RecR, and Ssb proteins resulted in the formation of RecF–RecO–Ssb complexes; i.e., RecR was excluded. Preincubation of RecF, RecO, and RecR proteins prior to addition of Ssb protein resulted in the formation of complexes consisting of RecF, RecO, RecR, and Ssb proteins. These data suggest that one role of RecF is to stabilize the interaction of RecR with RecO in the presence of Ssb protein. Finally, we found that interactions of RecF with RecO are lost in the presence of ATP. We discuss these results to explain how the RecF–RecO–RecR complex functions as an anti-Ssb factor.

HDA1 and RPD3 are members of distinct yeast histone deacetylase complexes that regulate silencing and transcription

Increased histone acetylation has been correlated with increased transcription, and regions of heterochromatin are generally hypoacetylated. In investigating the cause-and-effect relationship between histone acetylation and gene activity, we have characterized two yeast histone deacetylase complexes. Histone deacetylase-A (HDA) is an ≈350-kDa complex that is highly sensitive to the deacetylase inhibitor trichostatin A. Histone deacetylase-B (HDB) is an ≈600-kDa complex that is much less sensitive to trichostatin A. The HDA1 protein (a subunit of the HDA activity) shares sequence similarity to RPD3, a factor required for optimal transcription of certain yeast genes. RPD3 is associated with the HDB activity. HDA1 also shares similarity to three new open reading frames in yeast, designated HOS1, HOS2, and HOS3. We find that both hda1 and rpd3 deletions increase acetylation levels in vivo at all sites examined in both core histones H3 and H4, with rpd3 deletions having a greater impact on histone H4 lysine positions 5 and 12. Surprisingly, both hda1 and rpd3 deletions increase repression at telomeric loci, which resemble heterochromatin with rpd3 having a greater effect. In addition, rpd3 deletions retard full induction of the PHO5 promoter fused to the reporter lacZ. These data demonstrate that histone acetylation state has a role in regulating both heterochromatic silencing and regulated gene expression.

Mycoparasitic interaction relieves binding of the Cre1 carbon catabolite repressor protein to promoter sequences of the ech42 (endochitinase-encoding) gene in Trichoderma harzianum

The fungus Trichoderma harzianum is a potent mycoparasite of various plant pathogenic fungi. We have studied the molecular regulation of mycoparasitism in the host/mycoparasite system Botrytis cinerea/T. harzianum. Protein extracts, prepared from various stages of mycoparasitism, were used in electrophoretic mobility-shift assays (EMSAs) with two promoter fragments of the ech-42 (42-kDa endochitinase-encoding) gene of T. harzianum. This gene was chosen as a model because its expression is triggered during mycoparasitic interaction [Carsolio, C., Gutierrez, A., Jimenez, B., van Montagu, M. & Herrera-Estrella, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10903–10907]. All cell-free extracts formed high-molecular weight protein–DNA complexes, but those obtained from mycelia activated for mycoparasitic attack formed a complex with greater mobility. Competition experiments, using oligonucleotides containing functional and nonfunctional consensus sites for binding of the carbon catabolite repressor Cre1, provided evidence that the complex from nonmycoparasitic mycelia involves the binding of Cre1 to both fragments of the ech-42 promoter. The presence of two and three consensus sites for binding of Cre1 in the two ech-42 promoter fragments used is consistent with these findings. In contrast, the formation of the protein–DNA complex from mycoparasitic mycelia is unaffected by the addition of the competing oligonucleotides and hence does not involve Cre1. Addition of equal amounts of protein of cell-free extracts from nonmycoparasitic mycelia converted the mycoparasitic DNA–protein complex into the nonmycoparasitic complex. The addition of the purified Cre1::glutathione S-transferase protein to mycoparasitic cell-free extracts produced the same effect. These findings suggest that ech-42 expression in T. harzianum is regulated by (i) binding of Cre1 to two single sites in the ech-42 promoter, (ii) binding of a “mycoparasitic” protein–protein complex to the ech-42 promoter in vicinity of the Cre1 binding sites, and (iii) functional inactivation of Cre1 upon mycoparasitic interaction to enable the formation of the mycoparasitic protein–DNA complex.

Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants

A chimeric Lhcb gene encoding light-harvesting chlorophyll a/b-binding protein (LHCII) was expressed in transgenic tobacco plants. To separate native from recombinant LHCII, the protein was extended by six histidines at its C terminus. Recombinant LHCII was isolated by detergent-mediated monomerization of pure trimers followed by affinity-chromatography on Ni2+-NTA-agarose (NTA is nitrilotriacetic acid). Elution with imidazole yielded recombinant monomers that formed trimers readily after dilution of the detergent without further in vitro manipulations. LHCII subunits showed the typical chlorophyll a/b ratio at all steps of purification indicating no significant loss of pigments. Transgenic tobacco overexpressed amounts of recombinant protein that corresponded to about 0.7% of total LHCII. This yield suggested that expression in planta might be an alternative to the expression of eukaryotic membrane proteins in yeast. Recombinant LHCII was able to form two-dimensional crystals after addition of digalactolipids, which diffracted electrons to 3.6-Å resolution. LHCII carrying a replacement of Arg-21 with Gln accumulated to only 0.004% of total thylakoid proteins. This mutant was monomeric in the photosynthetic membrane probably due to the deletion of the phosphatidylglycerol binding site and was degraded by the plastidic proteolytic system. Exchange of Asn-183 with Leu impaired LHCII biogenesis in a similar way presumably due to the lack of a chlorophyll a binding site.

Immune response in human melanoma after transfer of an allogeneic class I major histocompatibility complex gene with DNA–liposome complexes

Analysis of the antitumor immune response after gene transfer of a foreign major histocompatibility complex class I protein, HLA-B7, was performed. Ten HLA-B7-negative patients with stage IV melanoma were treated in an effort to stimulate local tumor immunity. Plasmid DNA was detected within treated tumor nodules, and RNA encoding recombinant HLA-B7 or HLA-B7 protein was demonstrated in 9 of 10 patients. T cell migration into treated lesions was observed and tumor-infiltrating lymphocyte reactivity was enhanced in six of seven and two of two patients analyzed, respectively. In contrast, the frequency of cytotoxic T lymphocyte against autologous tumor in circulating peripheral blood lymphocytes was not altered significantly, suggesting that peripheral blood lymphocyte reactivity is not indicative of local tumor responsiveness. Local inhibition of tumor growth was detected after gene transfer in two patients, one of whom showed a partial remission. This patient subsequently received treatment with tumor-infiltrating lymphocytes derived from gene-modified tumor, with a complete regression of residual disease. Thus, gene transfer with DNA–liposome complexes encoding an allogeneic major histocompatibility complex protein stimulated local antitumor immune responses that facilitated the generation of effector cells for immunotherapy of cancer.

Involvement of protein kinase A in fibroblast growth factor-2-activated transcription

Polypeptide growth factors activate common signal transduction pathways, yet they can induce transcription of different target genes. The mechanisms that control this specificity are not completely understood. Recently, we have described a fibroblast growth factor (FGF)-inducible response element, FiRE, on the syndecan-1 gene. In NIH 3T3 cells, the FiRE is activated by FGF-2 but not by several other growth factors, such as platelet-derived growth factor or epidermal growth factor, suggesting that FGF-2 activates signaling pathways that diverge from pathways activated by other growth factors. In this paper, we report that the activation of FiRE by FGF-2 requires protein kinase A (PKA) in NIH 3T3 cells. The PKA-specific inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide) blocked the FGF-2-induced activation of FiRE, the transcription of the syndecan-1 gene, and cell proliferation. Also, expression of a dominant-negative form of PKA inhibited the FGF-2-induced FiRE activation and the transcription of the syndecan-1 gene. The binding of activator protein-1 transcription-factor complexes, required for the activation of FiRE, was blocked by inhibition of PKA activity before FGF-2 treatment. In accordance with the growth factor specificity of FiRE, the activity of PKA was stimulated by FGF-2 but not by platelet-derived growth factor or epidermal growth factor. Furthermore, a portion of the PKA catalytic subunit pool was translocated to the nucleus by FGF-2. Noticeably, the total cellular cAMP concentration was not affected by FGF-2 stimulus. We propose that the FGF-2-selective transcriptional activation through FiRE is caused by the ability of FGF-2 to control PKA activity.

GTP bound to chloroplast thylakoid membranes is required for light-induced, multienzyme degradation of the photosystem II D1 protein

Even though light is the driving force in photosynthesis, it also can be harmful to plants. The water-splitting photosystem II is the main target for this light stress, leading to inactivation of photosynthetic electron transport and photooxidative damage to its reaction center. The plant survives through an intricate repair mechanism involving proteolytic degradation and replacement of the photodamaged reaction center D1 protein. Based on experiments with isolated chloroplast thylakoid membranes and photosystem II core complexes, we report several aspects concerning the rapid turnover of the D1 protein. (i) The primary cleavage step is a GTP-dependent process, leading to accumulation of a 23-kDa N-terminal fragment. (ii) Proteolysis of the D1 protein is inhibited below basal levels by nonhydrolyzable GTP analogues and apyrase treatment, indicating the existence of endogenous GTP tightly bound to the thylakoid membrane. This possibility was corroborated by binding studies. (iii) The proteolysis of the 23-kDa primary degradation fragment (but not of the D1 protein) is an ATP- and zinc-dependent process. (iv) D1 protein degradation is a multienzyme event involving a strategic (primary) protease and a cleaning-up (secondary) protease. (v) The chloroplast FtsH protease is likely to be involved in the secondary degradation steps. Apart from its significance for understanding the repair of photoinhibition, the discovery of tightly bound GTP should have general implications for other regulatory reactions and signal transduction pathways associated with the photosynthetic membrane.

DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA–ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA–Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA–ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.

Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates

Point mutants of three unrelated antifluorescein antibodies were constructed to obtain nine different single-chain Fv fragments, whose on-rates, off-rates, and equilibrium binding affinities were determined in solution. Additionally, activation energies for unbinding were estimated from the temperature dependence of the off-rate in solution. Loading rate-dependent unbinding forces were determined for single molecules by atomic force microscopy, which extrapolated at zero force to a value close to the off-rate measured in solution, without any indication for multiple transition states. The measured unbinding forces of all nine mutants correlated well with the off-rate in solution, but not with the temperature dependence of the reaction, indicating that the same transition state must be crossed in spontaneous and forced unbinding and that the unbinding path under load cannot be too different from the one at zero force. The distance of the transition state from the ground state along the unbinding pathway is directly proportional to the barrier height, regardless of the details of the binding site, which most likely reflects the elasticity of the protein in the unbinding process. Atomic force microscopy thus can be a valuable tool for the characterization of solution properties of protein-ligand systems at the single molecule level, predicting relative off-rates, potentially of great value for combinatorial chemistry and biology.

Footprints on the viral DNA ends in Moloney murine leukemia virus preintegration complexes reflect a specific association with integrase

Retroviral DNA integration is mediated by the preintegration complex, a large nucleoprotein complex derived from the core of the infecting virion. We previously have used Mu-mediated PCR to probe the nucleoprotein organization of Moloney murine leukemia virus preintegration complexes. A region of protection spans several hundred base pairs at each end of the viral DNA, and strong enhancements are present near the termini. Here, we show that these footprints reflect a specific association between integrase and the viral DNA ends in functional preintegration complexes. Barrier-to-autointegration factor, a cellular protein that blocks autointegration of Moloney murine leukemia virus DNA, also plays an indirect role in generating the footprints at the ends of the viral DNA. We have exploited Mu-mediated PCR to examine the effect of mutations at the viral DNA termini on complex formation. We find that a replication competent mutant with a deletion at one end of the viral DNA still exhibits a strong enhancement about 20 bp from the terminus of the mutant DNA end. The site of the enhancement therefore appears to be at a fixed distance from the ends of the viral DNA. We also find that a mutation at one end of the viral DNA, which renders the virus incompetent for replication, abolishes the enhancements and protection at both the U3 and U5 ends. A pair of functional viral DNA ends therefore are required to interact before the chemical step of 3′ end processing.

Protein component of the ribozyme ribonuclease P alters substrate recognition by directly contacting precursor tRNA

The protein component of ribonuclease P (RNase P) binds to the RNA subunit, forming a functional ribonucleoprotein complex in vivo and enhancing the affinity of the precursor tRNA (pre-tRNA) substrate. Photocrosslinking experiments with pre-tRNA bound to RNase P reconstituted with the protein component of Bacillus subtilis ribonuclease P (P protein) site specifically modified with a crosslinking reagent indicate that: (i) the central cleft of P protein directly interacts with the single-stranded 5′ leader sequence of pre-tRNA, and (ii) the orientation and register of the pre-tRNA leader sequence in the central cleft places the protein component in close proximity to the active site. This unique mode of interaction suggests that the catalytic active site in RNase P occurs near the interface of RNA and protein. In contrast to other ribonucleoprotein complexes where the protein mainly stabilizes the active tertiary fold of the RNA, a critical function of the protein component of RNase P is to alter substrate specificity and enhance catalytic efficiency.