Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression

To define the current indications for surgical management of pleuropulmonary tuberculosis and analyze the results of operative procedures, the records of 59 patients operated on between January 1987 and December 1993 were reviewed. Three patient categories were defined. Group I patients (n = 25) underwent operation for diagnostic purposes: solitary mediastinal node or mediastinal adenopathy associated with pulmonary lesions (n = 10), pulmonary infiltrates (n = 4), pulmonary nodules or masses (n = 10), or chronic pleurisy (n = 1). Postoperative mortality and morbidity rates in this group were both 4%. Group II patients (n = 18) underwent operation for active lesions: intrapulmonary cavity (n = 6), destroyed lung parenchyma (n = 6), or chronic loculated pleural effusion (n = 6). Postoperative morbidity and mortality rates were 16.6% and 5.5%, respectively. Group III patients (n = 16) underwent operation for a complication of therapy or for sequelae of previously "cured" tuberculosis: calcified pyothorax (n = 8), empyema (n = 2), fistulized nodes (n = 2), bronchiectasis (n = 3), or aspergilloma (n = 1). Morbidity and mortality rates in this group were 31.25% and 12.5%, respectively. Surgery continues to have both diagnostic and therapeutic indications for management of pleuropulmonary tuberculosis, despite the morbidity and mortality rates associated with operative procedures.     

Biochemical properties and cellular localization of the Drosophila DG1 cGMP-dependent protein kinase

The protein encoded by the Drosophila cGMP-dependent protein kinase gene, DG1, was expressed in Sf9 cells. cGMP (10 microM) stimulated histone H2B phosphorylation by the DG1 protein kinase 20-fold. Maximal activity was observed at 40-50 mM Mg2+. The concentrations of cGMP, cAMP, cIMP, 8-bromo-cGMP, and 8-bromo-cAMP that gave 50% activation were 0.19 +/- 0.06, 11.7 +/- 2.8, 5.3 +/- 1.5, 0.04 +/- 0. 01, and 0.62 +/- 0.06 microM, respectively. cGMP activation was cooperative with a Hill coefficient (nH) of 1.28 +/- 0.10, whereas activation by cAMP was not cooperative. DG1 kinase expressed in Sf9 cells was found to be a dimer with an amino-terminal dimerization domain. It also autophosphorylated in a reaction stimulated by cGMP and cAMP. Immunoadsorbed DG1 protein from fly extracts was also capable of autophosphorylation, and this assay was used to quantitate the DG1 kinase in extracts from heads and bodies of adults and whole embryos. Activity was highest in heads of either sex and male bodies, intermediate in female bodies, and lowest in embryos. These results were in accord with DG1 mRNA abundance. Tissue distribution of the DG1 kinase was investigated by immunohistochemistry. In embryos, specific immunoreactivity was observed in large cells scattered along the anterior-posterior axis at stage 13. Prominent staining of adult heads was restricted to the proximal level of the lamina cortex.     

cGMP stimulation of cystic fibrosis transmembrane conductance regulator Cl- channels co-expressed with cGMP-dependent protein kinase type II but not type Ibeta

In order to investigate the involvement of cGMP-dependent protein kinase (cGK) type II in cGMP-provoked intestinal Cl- secretion, cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels was analyzed after expression of cGK II or cGK Ibeta in intact cells. An intestinal cell line which stably expresses CFTR (IEC-CF7) but contains no detectable endogenous cGK II was infected with a recombinant adenoviral vector containing the cGK II coding region (Ad-cGK II) resulting in co-expression of active cGK II. In these cells, CFTR was activated by membrane-permeant analogs of cGMP or by the cGMP-elevating hormone atrial natriuretic peptide as measured by 125I- efflux assays and whole-cell patch clamp analysis. In contrast, infection with recombinant adenoviruses expressing cGK Ibeta or luciferase did not convey cGMP sensitivity to CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only cGK II, infection with Ad-cGK II but not Ad-cGK Ibeta enabled cGMP analogs to increase CFTR phosphorylation in intact cells. These and other data provide evidence that endogenous cGK II is a key mediator of cGMP-provoked activation of CFTR in cells where both proteins are co-localized, e. g. intestinal epithelial cells. Furthermore, they demonstrate that neither the soluble cGK Ibeta nor cAMP-dependent protein kinase are able to substitute for cGK II in this cGMP-regulated function.     

The Salmonella typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG

The secretion of pathogenicity factors by Salmonella typhimurium is mediated by a type III secretion system that includes an outer membrane protein of the secretin family. Related secretins are also required for f1 phage assembly and type II secretion. When the C-terminal 43 amino acids of the S. typhimurium secretin InvG are added to f1 pIV, the chimeric f1 pIV-'InvG43 protein becomes dependent on the co-expression of another gene, invH, for function in phage assembly. [3H]-palmitic acid labelling, globomycin sensitivity and density gradient flotation were used to demonstrate that InvH is an outer membrane lipoprotein that is processed by signal peptidase II. A complex between chimeric f1 pIV-'InvG43 and InvH was demonstrated in vivo. InvH was shown to be required for the proper localization of InvG in the outer membrane and for the secretion of the virulence factor SipC. These results suggest that InvH and InvG are part of the functional outer membrane translocation complex in type III secretion systems.     

Arginine 75 in the pseudosubstrate sequence of type Ibeta cGMP-dependent protein kinase is critical for autoinhibition, although autophosphorylated serine 63 is outside this sequence

Autoinhibitory domains in many protein kinases include either a phosphorylatable substrate-like sequence or a pseudosubstrate sequence. This study shows that Ibeta cGMP-dependent protein kinase (cGK) autophosphorylates Ser-63, which is in an atypical cGK substrate sequence (-59AQKQAS-) that is amino-terminal to the pseudosubstrate motif (-74KRQAI-). cGMP increases the rate of autophosphorylation (approximately 0.8 phosphate/cGK monomer) approximately 3-fold. Autophosphorylation is an intramolecular process since it is independent of cGK concentration. cGMP activation of cGK enhances proteolysis within and near the pseudosubstrate site; treatment of dimeric cGK with three proteases produces three cGK monomers (approximately 67-70 kDa each). Their amino-terminal sequences are 75RQAISAEPT-, 76QAISAEPTAF-, and 86DIQDLSXV-, respectively. cGMP stimulates these kinases by 10-, 2.5-, and 1.4-fold, respectively, compared with a 10-fold effect on intact cGK. Increased basal activity accounts for the diminished stimulation. Thus, the primary autophosphorylation site of Ibeta cGK is well outside the pseudosubstrate site, but Arg-75 in the pseudosubstrate site is critical for autoinhibition. Autoinhibition also involves elements that are carboxyl-terminal to Arg-75.     

Herpes simplex virus 2 UL45 is a type II membrane protein

In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45.     

Phosphorylation and activation of cGMP-dependent protein kinase by Src

Naive CD8 T cells can be polarized into effectors producing the type 1 cytokines IFN-gamma and IL-2 or the type 2 cytokines IL-4, IL-5, and IL-10, respectively. To study whether the polarized cytokine phenotype of the effectors is stable, we generated highly cytotoxic hemagglutinin (HA) peptide-specific CD8 Tc1 and Tc2 (cytotoxic CD8 T cells producing type 1 or type 2 cytokines) effectors from Clone-4 TCR-transgenic mice, which were adoptively transferred into syngeneic adult thymectomized irradiated and bone marrow-reconstituted recipients. The highly activated blast-size, CD25+ Tc1 and Tc2 effectors gave rise to homogeneous resting CD25- CD44(high) Ly6C(high) Ag-specific populations, which persisted for at least 13 wk after adoptive transfer. These memory CD8 T cells, recovered 13 wk after transfer of Tc1 or Tc2 effectors, still produced either the type 1 or type 2 cytokines, i.e., IFN-gamma, or IL-4 and IL-5, respectively, upon restimulation with APCs loaded with the HA peptide, but not in the absence of Ag. The amounts of IL-2 detected in the supernatants of Tc1 and Tc2 memory populations were comparable to that in memory CD4 cells, and both Tc1 and Tc2 memory cells became cytotoxic upon restimulation. Thus, cytokine-polarized CD8 memory T cells are a source of a variety of cytokines, which were classically considered helper cytokines, opening new perspectives on their function as regulatory cells in an immune response.     

The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.     

Identification of cGMP-dependent protein kinase anchoring proteins (GKAPs)

To promote both efficiency and selectivity, many protein kinases and phosphatases are maintained in specific subcellular microenvironments through their association with anchoring proteins. In this study, we describe a new class of proteins, called GKAPS, that specifically bind the Type II cGMP-dependent protein kinase (PKG). GKAPs were detected in rat aorta, brain, and intestine using a protein overlay technique. The PKG binding proteins were distinct from AKAPs, proteins known to bind the cAMP-dependent protein kinase (PKA). Furthermore, a synthetic peptide that blocks association of PKA with AKAPs did not affect the PKG-GKAP interaction. Deletion mutagenesis was used to map the GKAP binding determinants within PKG to the N-terminal regulatory region. While most GKAPs were tissue-specific, a ubiquitous PKG-binding protein was detected and identified as myosin. Analysis of myosin fragments revealed that PKG binds within Subfragment 2. The results define a novel class of anchoring proteins that may target PKG for specific functional roles.     

Drosophila polo kinase is required for cytokinesis

A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.     

The Cdc7 protein kinase is required for origin firing during S phase

The Cdc7p protein kinase plays an essential, but undefined, role promoting S phase in the budding yeast, Saccharomyces cerevisiae. Previous experiments have shown that the essential function of Cdc7 is executed near the G1-S boundary; after Start but before the elongation phase of DNA replication. Origins of DNA replication fire throughout S phase in budding yeast. Therefore, the G1-S transition is a cell-cycle event that precedes, and is distinct from, the activation of individual origins. Consequently, we have asked whether Cdc7 is only required for S-phase entry or if it plays a role during S phase in origin firing. In this article, we show that partial loss of Cdc7 function results in slow progression through S phase rather than slow entry into S phase and that Cdc7 is still required for the timely completion of S phase after a block to elongation with hydroxyurea. This is because Cdc7 is still required for the activation of late-firing origins after the hydroxyurea block. These experiments show that, rather than acting as a global regulator of the G1-S transition, Cdc7 appears to play a more direct role in the firing of replication origins during S phase.     

Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A

Protein kinase A (PKA) stimulates Cl secretion by activating the cystic fibrosis transmembrane conductance regulator (CFTR), a tightly regulated Cl- channel in the apical membrane of many secretory epithelia. The CFTR channel is also modulated by protein kinase C (PKC), but the regulatory mechanisms are poorly understood. Here we present evidence that PKA-mediated phosphorylation alone is not a sufficient stimulus to open the CFTR chloride channel in the presence of MgATP; constitutive PKC phosphorylation is essential for acute activation of CFTR by PKA. When patches were excised from transfected Chinese hamster ovary cells, CFTR responses to PKA became progressively smaller with time and eventually disappeared. This decline in PKA responsiveness did not occur in the presence of exogenous PKC and was reversed by the addition of PKC to channels that had become refractory to PKA. PKC enhanced PKA stimulation of open probability without increasing the number of functional channels. Short-term pretreatment of cells with the PKC inhibitor chelerythrine (1 microM) reduced the channel activity that could be elicited by forskolin in cell-attached patches. Moreover, in whole cell patches, acute stimulation of CFTR currents by chlorophenylthio-cAMP was abolished by two chemically unrelated PKC inhibitors, although an abrupt, partial activation was observed after a delay of >15 min. Modulation by PKC was most pronounced when basal PKC phosphorylation was reduced by briefly preincubating cells with chelerythrine. Constitutive PKC phosphorylation in unstimulated cells permits the maximum elevation of open probability by PKA to reach a level that is approximately 60% of that attained during in vitro exposure to both kinases. Differences in basal PKC activity may contribute to the variable cAMP responsiveness of CFTR channels in different cell types.     

Mitogen-activated protein kinase is activated during IgG-mediated phagocytosis, but is not required for target ingestion

This meta-analysis quantitatively summarized the developmental influence and the effects of locomotor experience as well as the benefits of locomotor practice, locomotor assistance, and active searching patterns on children's search performance. Based on specific criteria, a search of a database and reference lists identified 19 studies, including 1,029 children (510 boys and 519 girls) from 4 to 144 months of age. Outcome measures of spatial performance were converted to 83 effect sizes that reflected the effects of specific experimental characteristics. Analyses of variance indicated that with older children, locomotor activities are more important to their spatial searching. Locomotor status, searching patterns, locomotor assistance, test conditions, and test reliability were identified as moderator variables. In addition, locomotor training significantly improved children's spatial search. The results supported the hypothesis that children's development of spatial search skills is influenced by locomotor experience.     

Nitric oxide modulates the c-Jun N-terminal kinase/stress-activated protein kinase activity through activating c-Jun N-terminal kinase kinase

Nitric oxide is a signaling molecule that has a broad range of physiological functions, including neurotransmission, macrophage activation, and vasodilation. The mechanism by which nitric oxide regulates signal transduction mediating diverse biological activities is not fully understood, however. Here, we demonstrate that nitric oxide induced the stimulation of c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK) in intact cells. Exposure of cultured HEK293 cells to sodium nitroprusside, a nitric oxide releasing agent, resulted in the stimulation of JNK1 activity. The sodium nitroprusside-induced stimulation of JNK1 activity was abolished by treatment of cells with N-acetylcysteine. Nitric oxide production from HEK293 cells ectopically expressing nitric oxide synthases resulted in the stimulation of JNK1 activity, while JNK1 stimulation in nitric oxide synthase-overexpressing cells was abrogated by a nitric oxide synthase inhibitor, NG-nitro-L-arginine. Furthermore, exposure of cells to sodium nitroprusside resulted in the stimulation of JNK kinase (JNKK1/SEK1). Taken together, our data suggest that nitric oxide modulates the JNK activity through activating JNKK1/SEK1.     

A C-terminal di-leucine is required for localization of the Menkes protein in the trans-Golgi network

The human X-linked recessive disorder of copper metabolism, Menkes disease, is caused by a defect in the MNK ( ATP7A ) gene which encodes a transmembrane copper-transporting P-type ATPase (MNK). MNK is an important component of the mammalian copper transport pathway, and previous studies in cultured cells have localized MNK to the final compartment of the Golgi apparatus, the trans -Golgi network (TGN). At this location, MNK is predicted to supply copper to copper-dependent enzymes as they migrate through the secretory pathway. However, under conditions of elevated extracellular copper, the MNK protein undergoes a rapid relocalization to the plasma membrane where it functions in the efflux of copper from cells. In this study, three di-leucine motifs and a cluster of four acidic amino acids within the C-terminal region of MNK were investigated as candidate signals necessary for steady-state TGN localization. In vitro mutagenesis of the human MNK cDNA and immunofluorescence detection of mutant forms of MNK expressed in cultured cells demonstrated that the di-leucine, L1487L1488, was essential for localization of MNK within the TGN, but not for copper efflux. We suggest that this di-leucine motif is a putative endocytic targeting motif necessary for the retrieval of MNK from the plasma membrane to the TGN. Our data, along with the recent demonstration that the third transmembrane region of MNK functions as a TGN targeting signal, suggests that MNK localization to the TGN may be a two-step process involving TGN retention via the transmembrane region, and recycling to this compartment from the plasma membrane via the L1487L1488 motif.     

Fission yeast wee1 protein kinase is not required for DNA damage-dependent mitotic arrest

Checkpoints maintain the dependency relationships between discrete events in the cell cycle (for example, ensuring mitosis does not occur before DNA replication is complete). In Schizosaccharomyces pombe, mitotic checkpoints monitor DNA synthesis and the presence of DNA damage. The replication-dependent mitotic checkpoint prevents mitosis by inactivating p34cdc2 kinase. The mechanism by which the DNA damage checkpoint interacts with the mitotic machinery is distinct from that used by the replication checkpoint. The activity of p34cdc2 is controlled, in part, by the wee1 protein kinase, which inactivates cdc2 through phosphorylation at tyrosine-15 (ref. 7). Here we report normal mitotic arrest after DNA damage in S. pombe cells in which the wee1 gene is defective or missing. We suggest why these findings contradict a recent report which suggested that the wee1 gene product was required for DNA damage-dependent mitotic arrest.     

The precore sequence of hepatitis B virus is required for nuclear localization of the core protein

The cellular localization of the precore/core and core proteins was studied by immunofluorescence following transfection of 143 thymidine kinase-negative (TK ) and Hep-G2 cells with expression constructs containing wild-type (hepatitis B e antigen [HBeAg]-positive) and precore mutant (HBeAg-negative) sequences. Precore/core constructs with the wild-type phenotype result in strong nuclear staining, while, in contrast, constructs expressing core antigen alone have strong cytoplasmic staining. These differences in the pattern of immunofluorescence staining may be caused by expression of the precore/core protein, some of which may be translocated into the nucleus, following removal of the signal peptide. In vitro translation experiments showed that the main protein products obtained in the presence of microsomal membranes were the precore/core protein and a truncated product representing the same protein without its signal peptide. Core protein expression from the precore mutant constructs was very much reduced, indicating that translational re-initiation was not very efficient. The significance of the precore/core protein being present in the nucleus is not clear, but suggests that it may be important in the replicative cycle of the virus. Finally, HBeAg produced by some of the constructs could not be detected because amino acid substitutions affected antibody-binding epitopes.     

Deletion mapping of N-terminal domains of surfactant protein A. The N-terminal segment is required for phospholipid aggregation and specific inhibition of surfactant secretion

The objective of the current study was to examine the functional importance of the N-terminal domains of surfactant protein A (SP-A) including the N-terminal segment from Asn1 to Ala7 (denoted domain 1), the N-terminal portion of the collagen domain from Gly8 to Gly44 (domain 2), and the C-terminal portion of the collagen-like domain from Gly45 to Pro80 (domain 3). Wild type recombinant SP-A (SP-Ahyp; where hyp indicates hydroxyproline-deficient) and truncated mutant (TM) SP-As containing deletions of domain(s) 1 (TM1), 2 (TM2), 1 and 2 (TM1-2), and 1, 2, and 3 (TM1-2-3) were synthesized in insect cells and purified by mannose-Sepharose affinity chromatography. N-terminal disulfide-dependent dimerization was preserved at near wild type levels in the TM1-2 (at Cys-1) and TM2 proteins (at Cys-1 and Cys6), and to a lesser extent in TM1 (at Cys-1), but not in TM1-2-3. Cross-linking analyses demonstrated that the neck + CRD was sufficient for assembly of monomers into noncovalent trimers and that the N-terminal segment was required for the association of trimers to form higher oligomers. All TM proteins except TM1-2-3 bound to phospholipid, but only the N-terminal segment containing TM proteins aggregated phospholipid vesicles. The TM1, TM1-2, and TM2 but not the TM1-2-3 inhibited the secretion of surfactant from type II cells as effectively as SP-Ahyp, but the inhibitory activity of each mutant was blocked by excess alpha-methylmannoside and therefore nonspecific. TM1 and TM1-2-3 did not enhance the uptake of phospholipids by isolated type II cells, but the TM1-2 and TM2 had activities that were 72 and 83% of SP-Ahyp, respectively. We conclude the following for SP-A: 1) trimerization does not require the collagen-like region or interchain disulfide linkage; 2) the N-terminal portion of the collagen-like domain is required for specific inhibition of surfactant secretion but not for binding to liposomes or for enhanced uptake of phospholipids into type II cells; 3) N-terminal interchain disulfide linkage can functionally replace the N-terminal segment for lipid binding, receptor binding, and enhancement of lipid uptake; 4) the N-terminal segment is required for the association of trimeric subunits into higher oligomers, for phospholipid aggregation, and for specific inhibition of surfactant secretion and cannot be functionally replaced by disulfide linkage alone for these activities.     

ER membrane protein complex required for nuclear fusion

Diploid cells of the yeast Saccharomyces cerevisiae form after the mating of two haploid cells of the opposite mating type. After fusion of the two plasma membranes of the mating cells, a dinucleated cell forms initially in which the two haploid nuclei then rapidly fuse to form a single diploid nucleus. This latter event, called karyogamy, can be divided into two distinct steps: the microtubule-based movement that causes the two nuclei to become closely juxtaposed and the fusion of the nuclear membranes. For the membrane fusion step, one required component, the ER luminal protein Kar2p (BiP), has been identified. For topological reasons, however, it has been unclear how Kar2p could function in this role. Kar2p is localized to the luminal (i.e., noncytoplasmic) face of the ER membrane, yet nuclear fusion must initiate from the cytosolic side of the outer nuclear membrane or the ER membrane with which it is contiguous. There is both genetic and biochemical evidence that Kar2p interacts with Sec63p, an ER membrane protein containing both luminal and cytosolic domains that is involved in protein translocation across the membrane. We have isolated novel sec63 mutant alleles that display severe karyogamy defects. Disruption of the genes encoding other Sec63p-associated proteins (Sec71p and Sec72p) also results in karyogamy defects. A suppressor mutant (sos1-1) partially corrects the translocation defect but does not alleviate the karyogamy defect. sec61 and sec62 mutant alleles that cause similar or more severe protein translocation defects show no karyogamy defects. Taken together, these results suggest a direct role for Sec63p, Sec71p, and Sec72p in nuclear membrane fusion and argue against the alternative interpretation that the karyogamy defects result as an indirect consequence of the impaired membrane translocation of another component(s) required for the process. We propose that an ER/nuclear membrane protein complex composed of Sec63p, Sec71p, and Sec72p plays a central role in mediating nuclear membrane fusion and requires ER luminally associated Kar2p for its function.     

Dimerization of Ste5, a mitogen-activated protein kinase cascade scaffold protein, is required for signal transduction

The mitogen-activated protein kinase cascade of the Saccharomyces cerevisiae pheromone response pathway is organized on the Ste5 protein, which binds each of the kinases of the cascade prior to signaling. In this study, a structure-function analysis of Ste5 deletion mutants uncovered new functional domains of the Ste5 protein and revealed that Ste5 dimerizes during the course of normal signal transduction. Dimerization, mediated by two regions in the N-terminal half of Ste5, was first suggested by intragenic complementation between pairs of nonfunctional Ste5 mutants and was confirmed by using the two-hybrid system. Coimmunoprecipitation of differently tagged forms of Ste5 from cells in which the pathway has been activated by Ste5 overexpression further confirmed dimerization. A precise correlation between the biological activity of various Ste5 fragments and dimerization suggests that dimerization is essential for Ste5 function.