Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.     

Phosphorylation triggers domain separation in the DNA binding response regulator NarL

DNA binding proteins of two-component signal transduction systems in microorganisms are activated by phosphorylation through an unknown mechanism. NarL is an example from the nitrate/nitrite signal transduction system of Escherichia coli. NarL consists of N- and C-terminal domains, the latter of which contains the DNA binding elements. To explore the mechanism of activation, single nitroxide side chains were introduced, one at a time, at nine different sites throughout the C-terminal domain to monitor the tertiary structure and the status of the surface in contact with the N-terminal domain. In addition, three pairs of doubly labeled proteins were prepared to monitor the interdomain distance using the magnetic dipolar interaction. The results of these site-directed spin-labeling studies reveal that phosphorylation at a distant site in the N-terminal domain triggers domain separation, likely by a hinge-bending motion. This in turn presents key elements of the C-terminal domain for docking to the DNA target in the configuration described in the recent crystal structure. The data also imply that a single conformation of unphosphorylated NarL exists in solution, and there is no detectable equilibrium between the closed and open conformations.     

t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast

The role of protein phosphorylation in secretion is not well understood. Here we show that yeast lacking the Snc1,2 v-SNAREs, or bearing a temperature-sensitive mutation in the Sso2 t-SNARE, are rescued at restrictive conditions by the addition of ceramide precursors and analogs to the growth medium. Rescue results from dephosphorylation of the Sso t-SNAREs by a ceramide-activated type 2A protein phosphatase (Sit4) involved in cell cycle control. Sso t-SNARE dephosphorylation correlated with its assembly into complexes with the Sec9 t-SNARE, both in vitro and in vivo, and with an increase in protein trafficking and secretion in cells. SNARE complexes isolated under these conditions contained only Sso and Sec9, suggesting that a t-t-SNARE fusion complex is sufficient to confer exocytosis. Mutation of a single PKA site (Ser79 to Ala79) in Sso1 resulted in a decrease in phosphorylation and was sufficient to confer growth to snc cells at restrictive conditions. Thus, modulation of t-SNARE phosphorylation regulates SNARE complex assembly and membrane fusion in vivo.     

Self-interaction of a SNARE transmembrane domain promotes the hemifusion-to-fusion transition

SNARE proteins mediate intracellular fusion of eukaryotic membranes. Some SNAREs have previously been shown to dimerise via interaction of their transmembrane domains. However, the functional significance of these interactions had remained unclear. Here, we show that mutating alternate faces of the transmembrane helix of the yeast vacuolar Q-SNARE Vam3p reduces the ability of the full-length protein to induce contents mixing in yeast vacuole fusion to different extents. Examination of liposome fusion induced by synthetic transmembrane domains revealed that inner leaflet mixing is delayed relative to outer leaflet mixing, suggesting that fusion transits through a hemifusion intermediate. Interestingly, one of the mutations impaired inner leaflet mixing in the liposome system. This suggests that the defect seen in vacuolar contents mixing is due to partial arrest of the reaction at hemifusion. Since covalent dimerisation of this mutant recovered wild-type behaviour, homodimerisation of a SNARE transmembrane domain appears to control the transition of a hemifusion intermediate to complete lipid mixing.     

Transmembrane organization of yeast syntaxin-analogue Sso1p

Membrane fusion in secretory pathways is thought to be mediated by SNAREs. It is proposed that membrane fusion transits through hemifusion, a condition in which the outer leaflets of the bilayers are mixed, but the inner leaflets are not. Hemifusion then proceeds to the fusion pore that connects the two internal contents. It is believed that the transmembrane domains (TMDs) of the fusion proteins play an essential role in the transition from hemifusion to the fusion pore. In this work, the structure, dynamics, and membrane topology of the TMD of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast, was investigated using site-directed spin labeling and EPR spectroscopy. The EPR analysis of spin-labeled mutants showed that the TMD of Sso1p is a well-defined membrane spanning alpha-helix. The results also indicate that there is an equilibrium between the monomers and the oligomers. The oligomerization is mainly mediated through the interaction at the N-terminal half of the TMD, whereas the C-terminal half is free of the tertiary interaction. Additionally, the isotropic hyperfine splitting values were examined for nitroxide-scanning mutants, and it was found that the hyperfine splitting values show a V-shaped profile across the bilayer. Thus, hyperfine splitting may be used as an additional parameter to measure bilayer immersion depths of nitroxide.     

The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo

Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.     

Phosphorylation of PDZ1 domain attenuates NHERF-1 binding to cellular targets

NHERF-1 (Na(+)-H(+) exchanger regulatory factor 1, also known as EBP50 ezrin-binding protein of 50 kDa) is a phosphoprotein that assembles multiprotein complexes via two PDZ domains and a C-terminal ezrin-binding domain. Current work utilized metabolic labeling in cultured cells expressing wild type GFP-NHERF-1 to define the physiological importance of NHERF-1 phosphorylation. Treatment of cells with phosphatase inhibitors calyculin A and okadaic acid enhanced NHERF-1 phosphorylation and inhibited its dimerization. Eliminating C-terminal serines abolished the modulation of NHERF-1 dimerization by phosphatase inhibitors and identified the phosphorylation of the PDZ1 domain that attenuated its binding to physiological targets, including beta(2)-adrenergic receptor, platelet-derived growth factor receptor, cystic fibrosis transmembrane conductance regulator, and sodium-phosphate cotransporter type IIa. The major covalent modification of PDZ1 was mapped to serine 77. Confocal microscopy of cultured cells suggested key roles for PDZ1 and ERM-binding domain in localizing NHERF-1 at the cell surface. The substitution S77A eliminated PDZ1 phosphorylation and increased NHERF-1 localization at the cell periphery. In contrast, S77D reduced NHERF-1 colocalization with cortical actin cytoskeleton. These data suggested that serine 77 phosphorylation played key role in modulating NHERF-1 association with plasma membrane targets and identified a novel mechanism by which PDZ1 phosphorylation may transduce hormonal signals to regulate the function of membrane proteins in epithelial tissues.     

Identification of an autoinhibitory domain in calcineurin

The hypothesis that calcineurin, the Ca2+/calmodulin-dependent protein phosphatase, contains an autoinhibitory domain was tested using synthetic peptides corresponding to regions of the carboxyl-terminus of calcineurin. Of the several peptides analyzed, one, containing residues I-T-S-F-E-E-A-K-G-L-D-R-I-N-E-R-M-P-P-R-R-D-A-M-P, gave complete inhibition of its protein phosphatase activity. Using [32P]myosin light chain as substrate an IC50 of about 10 microM was obtained with either native calcineurin, assayed in the presence of Ca2+/calmodulin, or with calcineurin subjected to partial proteolysis which converts it to a fully active phosphatase when assayed in the presence of [ethylenebis (oxyethylenenitrilo)]tetraacetic acid. With 50 mM p-nitrophenylphosphate as substrate an IC50 of about 40 microM was observed. Studies with overlapping peptides suggested that the sequence P-P-R-R-D-A-M-P was essential but not sufficient for the observed inhibition. Kinetic analysis indicated that the inhibition of phosphatase activity was not competitive with respect to [32P]myosin light chain. This peptide did not show significant inhibition of the catalytic subunits of protein phosphatases type I or type IIA or of Ca2+/calmodulin-dependent protein kinase II. These results indicate that amino acids within this sequence of calcineurin constitute a unique autoinhibitory domain which interacts with the active site and is responsible for the low basal phosphatase activity in the absence of Ca2+/calmodulin.     

Regulation of WNK1 by an autoinhibitory domain and autophosphorylation

WNK family protein kinases are large enzymes that contain the catalytic lysine in a unique position compared with all other protein kinases. These enzymes have been linked to a genetically defined form of hypertension. In this study we introduced mutations to test hypotheses about the position of the catalytic lysine, and we examined mechanisms involved in the regulation of WNK1 activity. Through the analysis of enzyme fragments and sequence alignments, we have identified an autoinhibitory domain of WNK1. This isolated domain, conserved in all four WNKs, suppressed the activity of the WNK1 kinase domain. Mutation of two key residues in this autoinhibitory domain attenuated its ability to inhibit WNK kinase activity. Consistent with these results, the same mutations in a WNK1 fragment that contain the autoinhibitory domain increased its kinase activity. We also found that WNK1 expressed in bacteria is autophosphorylated; autophosphorylation on serine 382 in the activation loop is required for its activity.     

Phosphorylation and intramolecular stabilization of the ligand binding domain in the nuclear receptor steroidogenic factor 1

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N'-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors.     

Characterisation of the DNA binding domain of the yeast RAP1 protein.

The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'helix-turn-helix' structural motifs. All the RAP1 binding sites we have tested bind domain 361-596, arguing that RAP1 binds all its chromosomal sites via this domain. The domain could not be further reduced in size suggesting that it represents the minimal functional DNA binding domain. The relevance of potential regions of secondary structure within the minimal binding domain is discussed.     

Regulation of MTK1/MEKK4 kinase activity by its N-terminal autoinhibitory domain and GADD45 binding

A variety of cellular stresses activate the stress-responsive mitogen-activated protein (MAP) kinases p38 and JNK. In this study, we studied the activation mechanism of a human MAP kinase kinase kinase, MTK1 (also known as MEKK4), which mediates activation of both p38 and JNK. MTK1 has an extensive N-terminal noncatalytic domain composed of approximately 1,300 amino acids. Full-length or near full-length MTK1 is catalytically inactive when expressed in Saccharomyces cerevisiae cells, as it is in mammalian cells. Deletion of a segment including positions 253 to 553 activates kinase, indicating that this segment contains the autoinhibitory domain. In the autoinhibited conformation, the MTK1 kinase domain cannot interact with its substrate, MKK6. By a functional complementation screening with yeast cells, GADD45 proteins (GADD45alpha, beta, and gamma) were identified as MTK1 activators. GADD45 proteins bind a site in MTK1 near the inhibitory domain and relieve autoinhibition. Mutants of full-length MTK1 were isolated that can interact with MKK6 in the absence of the activator GADD45 proteins. These MTK1 mutants are constitutively active, in both yeast and mammalian cells. A model of MTK1 autoinhibition by the N-terminal inhibitory domain and activation by GADD45 binding is presented.     

Phosphorylation of the integrin alpha 4 cytoplasmic domain regulates paxillin binding

alpha4 integrins are essential for embryogenesis, hematopoiesis, inflammation, and immune response possibly because alpha4 integrins have distinct signaling properties from other integrins. Specifically, the alpha4 cytoplasmic domain binds tightly to paxillin, a signaling adaptor protein, leading to increased cell migration and an altered cytoskeletal organization that results in reduced cell spreading. The alpha4 tail contains potential phosphorylation sites clustered in its core paxillin binding region. We now report that the alpha4 tail is phosphorylated in vitro and in vivo. Furthermore, Ser(988) is a major phosphorylation site. Using antibodies specific for Ser(988)-phosphorylated alpha4, we found the stoichiometry of alpha4 phosphorylation varied in different cells. However, >60% of alpha4 was phosphorylated in Jurkat T cells. Phosphorylation at Ser(988) blocked paxillin binding to the alpha4 tail. A phosphorylation-mimicking mutant of alpha4 (alpha4S988D) blocked paxillin binding and reversed the inhibitory effect of alpha4 on cell spreading. Consequently, alpha4 phosphorylation is a biochemical mechanism to modulate paxillin binding to alpha4 integrins with consequent regulation of alpha4 integrin-dependent cellular functions.     

Phosphorylation of Caldesmon by PFTAIRE1 kinase promotes actin binding and formation of stress fibers

Caldesmon (CaD) is an actin-binding protein that is capable of stabilizing actin filaments. Phosphorylation of CaD is widely accepted in the actin cytoskeletal modeling and promotion of cell migration. In this study, we show that CaD is a downstream phosphorylation substrate of PFTK1, a novel Cdc-2-related ser/thr protein kinase. Our study stemmed from an earlier investigation where we demonstrated that PFTK1 kinase conferred cell migratory advantages in human hepatocellular carcinoma (HCC) cells. Here, we showed that PFTK1-knockdown cells exhibited much reduced CaD phosphorylation and consequently caused dissociation of CaD from the F-actin fibers. The cellular localization of CaD was also altered in the absence of PFTK1. Immunofluorescence analysis revealed that PFTK1-abrogated cells exhibited a diffused and blurred appearance of CaD localization, whereas intact co-localization with F-actins was apparent in PFTK1-expressing cells. Without the binding of CaD to actin, disappearance of actin stress fibers was also evident in PFTK1-abrogated cells. In addition, we found that CaD is also commonly up-regulated in HCC tumors when compared to adjacent non-malignant liver (P = 0.022). Taken together, our results highlight a novel biological cascade that involved the phosphorylation activation of CaD by PFTK1 kinase in promoting formation of actin stress fibers.     

UvrB domain 4, an autoinhibitory gate for regulation of DNA binding and ATPase activity

UvrB, a central DNA damage recognition protein in bacterial nucleotide excision repair, has weak affinity for DNA, and its ATPase activity is activated by UvrA and damaged DNA. Regulation of DNA binding and ATP hydrolysis by UvrB is poorly understood. Using atomic force microscopy and biochemical assays, we found that truncation of domain 4 of Bacillus caldotenax UvrB (UvrBDelta4) leads to multiple changes in protein function. Protein dimerization decreases with an approximately 8-fold increase of the equilibrium dissociation constant and an increase in DNA binding. Loss of domain 4 causes the DNA binding mode of UvrB to change from dimer to monomer, and affinity increases with the apparent dissociation constants on nondamaged and damaged single-stranded DNA decreasing 22- and 14-fold, respectively. ATPase activity by UvrBDelta4 increases 14- and 9-fold with and without single-stranded DNA, respectively, and UvrBDelta4 supports UvrA-independent damage-specific incision by Cho on a bubble DNA substrate. We propose that other than its previously discovered role in regulating protein-protein interactions, domain 4 is an autoinhibitory domain regulating the DNA binding and ATPase activities of UvrB.     

Mso1 is a novel component of the yeast exocytic SNARE complex

The yeast exocytic SNARE complex consists of one molecule each of the Sso1/2 target SNAREs, Snc1/2 vesicular SNAREs, and the Sec9 target SNARE, which form a fusion complex that is conserved in evolution. Another protein, Sec1, binds to the SNARE complex to facilitate assembly. We show that Mso1, a Sec1-interacting protein, also binds to the SNARE complex and plays a role in mediating Sec1 functions. Like Sec1, Mso1 bound to SNAREs in cells containing SNARE complexes (i.e. wild-type, sec1-1, and sec18-1 cells), but not in cells in which complex formation is inhibited (i.e. sec4-8 cells). Nevertheless, Mso1 remained associated with Sec1 even in sec4-8 cells, indicating that they act as a pair. Mso1 localized primarily to the plasma membrane of the bud when SNARE complex formation was not impaired but was mostly in the cytoplasm when assembly was prevented. Genetic studies suggest that Mso1 enhances Sec1 function while attenuating Sec4 GTPase function. This dual action may impart temporal regulation between Sec4 turnoff and Sec1-mediated SNARE assembly. Notably, a small region at the C terminus of Mso1 is conserved in the mammalian Munc13/Mint proteins and is necessary for proper membrane localization. Overexpression of Mso1 lacking this domain (Mso1-(1-193)) inhibited the growth of cells bearing an attenuated Sec4 GTPase. These results suggest that Mso1 is a component of the exocytic SNARE complex and a possible ortholog of the Munc13/Mint proteins.