An autoinhibitory mechanism for nonsyntaxin SNARE proteins revealed by the structure of Ykt6p.

Ykt6p is a nonsyntaxin SNARE implicated in multiple intracellular membrane trafficking steps. Here we present the structure of the NH2-terminal domain of Ykt6p (Ykt6pN, residues 1 to 140). The structure of Ykt6pN differed entirely from that of syntaxin and resembled the overall fold of the actin regulatory protein, profilin. Like some syntaxins, Ykt6p adopted a folded back conformation in which Ykt6pN bound to its COOH-terminal core domain. The NH2-terminal domain plays an important biological role in the function of Ykt6p, which in vitro studies revealed to include influencing the kinetics and proper assembly of SNARE complexes.

AtBS14a and AtBS14b, two Bet1/Sft1-like SNAREs from Arabidopsis thaliana that complement mutations in the yeast SFT1 gene.

SNAREs are membrane-associated proteins that play a central role in vesicle targeting and intra-cellular membrane fusion reactions in eukaryotic cells. Here we describe the identification of AtBS14a and AtBS14b, putative SNAREs from Arabidopsis thaliana that share 60% amino acid sequence identity. Both AtBS14a and BS14b are dosage suppressors of the temperature-sensitive growth defect in sft1-1 cells and over-expression of either AtBS14a or AtBS14b can support the growth of sft1Delta cells but not bet1Delta cells. These data together with structure-function and biochemical studies presented herein suggest that AtBS14a and AtBS14b share properties that are consistent with them being members of the Bet1/Sft1 SNARE protein family.

Selective formation of Sed5p-containing SNARE complexes is mediated by combinatorial binding interactions.

Sed5p is the only syntaxin family member required for protein transport through the yeast Golgi and it is known to bind up to nine other soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins in vivo. We describe in vitro binding experiments in which we identify ternary and quaternary Sed5p-containing SNARE complexes. The formation of SNARE complexes among these endoplasmic reticulum- and Golgi-localized proteins requires Sed5p and is syntaxin-selective. In addition, Sed5p-containing SNARE complexes form selectively and this selectivity is mediated by Sed5p-containing intermediates that discriminate among subsequent binding partners. Although many of these SNAREs have overlapping distributions in vivo, the SNAREs that form complexes with Sed5p in vitro reflect their functionally distinct locales. Although SNARE-SNARE interactions are promiscuous and a single SNARE protein is often found in more than one complex, both the biochemical as well as genetic analyses reported here suggest that this is not a result of nonselective direct substitution of one SNARE for another. Rather our data are consistent with the existence of multiple (perhaps parallel) trafficking pathways where Sed5p-containing SNARE complexes play overlapping and/or distinct functional roles.

Yeast Golgi SNARE interactions are promiscuous.

The transport of proteins between various compartments of the secretory pathway occurs by the budding of vesicles from one membrane and their fusion with another. A key event in this process is the selective recognition of the target membrane by the vesicle and the current view is that SNARE protein interactions likely play a central role in vesicle-target recognition and or membrane fusion. In yeast, only a single syntaxin (Sed5p) is required for Golgi transport and Sed5p is known to bind to at least 7 SNARE proteins. However, the number of Sed5p-containing SNARE complexes that exist in cells is not known. In this study we examined direct pair-wise interactions between full length soluble recombinant forms of SNAREs (Sed5p, Sft1p, Ykt6p, Vti1p, Gos1p, Sec22p, Bos1p, and Bet1p) involved in ER-Golgi and intra-Golgi membrane trafficking. In the binding assay that we describe here the majority of SNARE-binary interactions tested were positive, indicating that SNARE-SNARE interactions although promiscuous are not entirely non-selective. Interactions between a number of the genes encoding these SNAREs are consistent with our binding data and taken together our results suggest that functionally redundant Golgi SNARE-complexes exist in yeast. In particular, over-expression of Bet1p (a SNARE required for ER-Golgi and Golgi-ER traffic) and can bypass the requirement for the otherwise essential SNARE Sft1p (required for intra-Golgi traffic), suggesting that Bet1p either functions in a parallel pathway with Sft1p or can be incorporated into SNARE-complexes in place of Sftp1. None-the-less this result suggests that Bet1p can participate in two distinct trafficking steps, cycling between the ER and Golgi as well as in retrograde intra-Golgi traffic. In addition, suppressor genetics together with the analysis of the phenotypes of conditional mutations in Sft1p and Ykt6p, are consistent with a role for these SNAREs in more than one trafficking step. We propose that different combinations of SNAREs form complexes with Sed5p and are required for multiple steps in ER-Golgi and intra-Golgi vesicular traffic. And that the apparent promiscuity of SNARE-SNARE binding interactions, together with the requirement for some SNAREs in more than one trafficking step, supports the view that the specificity of vesicle fusion events cannot be explained solely on the basis of SNARE-SNARE interactions.

A SNARE-like protein required for traffic through the Golgi complex.

The secretory pathway of eukaryotic cells comprises several distinct membrane-bound compartments which are interconnected by transport vesicles that pinch off from one membrane and fuse with the next. Targeting of these vesicles is mediated in part by interactions between integral membrane proteins on the vesicles and target organelles (soluble NSF attachment protein receptors (SNAREs)), termed v-SNAREs and t-SNAREs, respectively. SNAREs required for endoplasmic reticulum (ER)-Golgi transport and for fusion of vesicles with the plasma membrane are already known. Here we identify two yeast membrane proteins that show genetic interactions with Sed5p, which is the t-SNARE for ER-Golgi traffic. One of these membrane proteins, Sft1p, is structurally similar to the known v-SNAREs and is required for transport from an early to a later Golgi compartment. Our results indicate that a single t-SNARE can control more than one transport step, and provide the first candidate for a SNARE involved in intra-Golgi traffic.

Localization of Sed5, a putative vesicle targeting molecule, to the cis-Golgi network involves both its transmembrane and cytoplasmic domains.

The yeast Sed5 protein, which is required for vesicular transport between ER and Golgi complex, is a membrane protein of the syntaxin family. These proteins are thought to provide the specific targets that are recognized by transport vesicles. We have investigated the mechanism by which Sed5 protein is itself localized. Expression of epitope-tagged versions of the yeast, Drosophila and rat Sed5 homologues in COS cells results in a perinuclear distribution; immuno-EM reveals that the majority of the protein is in a tubulo-vesicular compartment on the cis side of the Golgi apparatus. A similar distribution was obtained with a chimeric molecule consisting of a plasma membrane syntaxin with the Drosophila Sed5 transmembrane domain. This indicates that the membrane-spanning domain contains targeting information, as is the case with resident Golgi enzymes. However, alterations to the transmembrane domain of Drosophila Sed5 itself did not result in its mistargeting, implying that an additional targeting mechanism exists which involves only the cytoplasmic part of the protein. This was confirmed by modifying the transmembrane domain of the yeast Sed5 protein: substitution with the corresponding region from the Sso1 protein (a plasma membrane syntaxin homologue) did not affect yeast Sed5 function in vivo.

Characterization of a stable form of human meizothrombin derived from recombinant prothrombin (R155A, R271A, and R284A).

Meizothrombin is a transient intermediate produced during the activation of prothrombin by the prothrombinase complex. Because meizothrombin is very sensitive to further activation and autolysis, its isolation is possible only in the presence of active site thrombin inhibitors. This complicates studies of the activities and functions of meizothrombin. As a model, we have expressed a mutant human prothrombin cDNA (R155A, R271A, R284A) with three of the cleavage sites modified so that they are no longer cleaved by factor Xa or thrombin. Several stable baby hamster kidney cell lines were isolated that secreted up to 20 micrograms/ml of carboxylated mutant prothrombin. After purification, the mutant prothrombin was activated by the prothrombinase complex or by ecarin, resulting in the formation of a meizothrombin-like molecule. Electrophoretic analysis and NH2-terminal sequence analysis were consistent with cleavage of a single bond between Arg320-Ile321 and proper processing of the prepropeptide. The meizothrombin was stable for weeks at 4 degrees C. Activation in the presence of dansylarginine N-(3-ethyl-1,5-pentanediyl) amide confirmed the conversion of prothrombin via meizothrombin. Compared with human plasma-derived thrombin, recombinant meizothrombin demonstrated approximately 7% clotting activity, 100% p-toluene-sulfonylarginine methyl ester esterase activity, and approximately 35% S2238 amidolytic activity, and could attenuate fibrinolysis.

Evolution of prothrombin: isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin.

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.

Characterization of wild-type and an active-site mutant in Escherichia coli of short-chain acyl-CoA dehydrogenase from Megasphaera elsdenii.

The objective of this work is to determine the molecular mechanism and regulation of short-chain acyl-CoA dehydrogenase (SCAD) from Megasphaera elsdenii. To achieve this, the gene coding for SCAD from M. elsdenii was cloned and sequenced. Site-directed mutagenesis was then used to identify an amino acid residue that is required for the proposed mechanism. To clone the gene, the amino acid sequence of the 50 N-terminal residues of SCAD from M. elsdenii was determined. This sequence information was utilized to synthesize two sets of mixed oligonucleotide primers which were then used to generate a 120-bp specific probe from M. elsdenii DNA by the polymerase chain reaction (PCR) method. The 120-bp probe was used to screen a M. elsdenii genomic DNA library cloned into Escherichia coli. The gene encoding M. elsdenii SCAD was identified from this library, sequenced, and expressed. The cloned SCAD gene contained an open reading frame which revealed a high degree of sequence identity with an open reading frame protein sequence of the human SCAD and the rat medium-chain acyl-CoA dehydrogenase (MCAD) (44% and 36% identical residues in paired comparisons for human SCAD and rat MCAD, respectively). Recombinant SCAD expressed from a pUC119 vector accounted for 35% of the cytosolic protein in the Escherichia coli crude extract. The expressed protein had similar activity, redox potential properties, and nearly identical amino acid composition to native M. elsdenii SCAD. In addition, a site-directed Glu367 Gln mutant of SCAD expressed from a pUC119 vector was shown to have minimal reductive and oxidative pathway activity with butyryl-CoA and crotonyl-CoA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of glycosylated and nonglycosylated human transferrin in mammalian cells. Characterization of the recombinant proteins with comparison to three commercially available transferrins.

The coding sequence for human serum transferrin was assembled from restriction fragments derived from a full-length cDNA clone isolated from a human liver cDNA library. The assembled clone was inserted into the expression vector pNUT and stably transfected into transformed baby hamster kidney (BHK) cells, leading to secretion of up to 125 mg/L recombinant protein into the tissue culture medium. As judged by mobility on NaDodSO4-PAGE, immunoreactivity, spectral properties (indicative of correct folding and iron binding), and the ability to bind to receptors on a human cell line, initial studies showed that the recombinant transferrin, is identical to three commercial human serum transferrin samples. Electrospray mass spectrometry (ESMS), anion-exchange chromatography, and urea gel analysis showed that the recombinant protein has an extremely complex carbohydrate pattern with 16 separate masses ranging from 78,833 to 80,802 daltons. Mutation of the two asparagine carbohydrate linkage sites to aspartic acid residues led to the expression and secretion of up to 25 mg/L nonglycosylated transferrin. ESMS, anion-exchange chromatography, and urea gel analysis showed a single molecular species that was consistent with the expected theoretical mass of 75,143 daltons. In equilibrium binding experiments, the nonglycosylated mutant bound to HeLa S3 cells with the same avidity and to the same extent as the glycosylated protein and the three commercial samples. These studies demonstrate conclusively that carbohydrate has no role in this function.

Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.

We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels.

The isolation and characterization of rabbit motilin precursor cDNA.

The cDNA sequence of rabbit motilin precursor has been determined. The predicted amino acid sequence indicates that the precursor consists of 133 amino acids and includes a 25 amino acid signal peptide followed by the 22 amino acid motilin sequence and an 86 amino acid motilin associated peptide (MAP). As in the human and porcine precursors, two lysine residues follow motilin in the rabbit sequence. Rabbit motilin shares 64% amino acid sequence identity with human and porcine motilin, and all amino acid substitutions represent conservative changes. Amino acid sequence alignments of the rabbit, human and porcine MAP sequences suggest three functional/structural motifs corresponding to a putative endoproteinase recognition site, a putative PEST site and a potential posttranslational processing recognition element.

Partial characterization of vertebrate prothrombin cDNAs: amplification and sequence analysis of the B chain of thrombin from nine different species.

The cDNA sequence of the B chain of thrombin (EC 3.4.21.5) has been determined from nine vertebrate species (rat, mouse, rabbit, chicken, gecko, newt, rainbow trout, sturgeon, and hagfish). The amino acid sequence identities vary from 96.5% (rat vs. mouse) to 62.6% (newt vs. hagfish). Of the 240 amino acids spanned in all the species compared, there is identity at 110 (45.8%) positions. When conservative changes are included, the amino acid similarity increases to 75%. The most conserved portions of the B chain are the active-site residues and adjacent amino acids, the B loop, and the primary substrate-binding region. In addition, the Arg-Gly-Asp motif is conserved in 9 of the 11 species compared, and the chemotactic/growth factor domain is well conserved in all of the 11 species compared. The least conserved regions of the B chain correspond to surface loops, including the putative thrombomodulin-binding sites and one of the hirudin-binding regions. The extent of the amino acid sequence similarity and the conservation of many of the functional/structural motifs suggests that, in addition to their role in blood coagulation, vertebrate thrombins may also play an important role in the general mechanisms of wound repair.

The nucleotide sequence of rabbit liver transferrin cDNA.

The cDNA sequence of rabbit liver transferrin has been determined. The largest cDNA was 2279 base pairs (bp) in size and encoded 694 amino acids consisting of a putative 19 amino acid signal peptide and 675 amino acids of plasma transferrin. The deduced amino acid sequence of rabbit liver transferrin shares 78.5% identity with human liver transferrin and 69.1% and 44.8% identity with porcine and Xenopus transferrins, respectively. At the amino acid level, vertebrate transferrins share 26.4% identity and 56.5% similarity. The most conserved regions correspond to the iron ligands and the anion binding region. Optimal alignment of transferrin sequences required the insertion of a number of gaps in the region corresponding to the N-lobe. In addition, the N-lobes of transferrins share less amino acid sequence similarity than the C-lobes.

Apolipoprotein E genotyping using the polymerase chain reaction and allele-specific oligonucleotide primers.

A method for apolipoprotein (apo) E genotyping was developed using the polymerase chain reaction (PCR) with allele-specific oligonucleotide primers (ASP). Synthetic oligonucleotides with base-pair mismatches at the 3' terminus were used as primers to amplify the apoE gene in subjects previously phenotyped using isoelectric focusing (IEF). Complementary primer-allele combinations were specifically amplified by PCR, together with a control pair of primers specific to the human prothrombin gene. Identification of genotype by PCR using ASP was consistent with the phenotypes that were determined by IEF for 14 healthy normolipidemic subjects. These results were achieved using DNA isolated from buccal epithelial cells obtained from a mouthwash or DNA extracted from leukocytes. Genotype identification required analysis of the PCR products on an ethidium-stained agarose gel, yielding results 3 h after DNA extraction. In comparison with other current methods, PCR using ASP is suggested as a rapid and simple noninvasive technique for determining population apoE allelic distribution.

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea star Pisaster ochraceus.

We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea star Pisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA(glu) and tRNA(thr) are 3' to 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.