Docking of ER‐derived vesicles to the cis‐Golgi compartment in yeast requires vesicle and target membrane receptors (v‐SNAREs and t‐SNAREs) and the GTPase Ypt1p. The t‐SNARE Sed5p is complexed with Sly1p in vivo. The mutant form Sly1‐20p rescues Ypt1p‐lacking cells from lethality, suggesting an inhibitory function of Sly1p in v‐SNARE/t‐SNARE interaction. Using surface plasmon resonance spectroscopy, we found that Sed5p binds Sly1p and Sly1‐20p with equally high affinity (K
D=5.13×10−9 M and 4.74×10−9 M, respectively). Deletion studies show that the N‐terminal half of Sly1p rather than the C‐terminus (harbouring the E532K substitution in Sly1‐20p) is most critical for its binding to Sed5p. These data appear to argue for an active rather than an inhibitory role of Sly1p in vesicle docking.
By complementation of a salt-sensitive mutant of Saccharomyces cerevisiae, we cloned the SOP1 gene, encoding a 114.5-kDa protein of 1033 amino acids. Cells deleted for SOP1 exhibited sensitivity to sodium stress, but showed no sensitivity to general osmotic stress. Following exposure of sop1⌬ cells to NaCl stress, the intracellular Na ؉ level and the Na ؉ /K ؉ ratio rose to values significantly higher than in wild type cells. Deletion of SOP2, encoding a protein sharing 54% amino acid identity with Sop1p, produced only slight Na ؉ sensitivity. Cells carrying a sop1⌬sop2⌬ double deletion became, however, hypersensitive to Na ؉ and exhibited increased sensitivity also to Li ؉ and K ؉ , suggesting involvement of both SOP1 and SOP2 in cation homeostasis. The predicted amino acid sequences of Sop1p and Sop2p show significant homologies with the cytoskeletal-associated protein encoded by the Drosophila lethal(2)giant larvae tumor suppressor gene. Immunolocalization of Sop1p revealed a cytoplasmic distribution and cell fractionation studies showed that a significant fraction of Sop1p was recovered in a sedimentable fraction of the cytosolic material. Expression of a Drosophila l(2)gl cDNA in the sop1⌬sop2⌬ strain partially restored the Na ؉ tolerance of the cells, indicating a functional relationship between the Sop proteins and the tumor suppressor protein, and a novel function in cell homeostasis for this family of proteins extending from yeast to human.
L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest K , = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (K, = 22 mM) was the only other substrate. V/K, for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 pkat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (a, 30 kDa; p, 25 kDa). The molecular mass of the native enzyme was estimated as 200 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine ( 5 mM each). However, by exposure t o air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 f 0.2 mol Fe and 5.6 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.The enzyme L-serine dehydratase catalyzes the overall deamination of L-serine to pyruvate. The initial step is a pelimination of water followed by tautomerization and hydrolysis to pyruvate and ammonia [l]. L-Serine dehydratases as well as the related threonine dehydratases are ubiquitous enzymes found in high amounts in mammalian liver [2], Saccharomyces cerevisiae [3,4] and in a variety of eubacteria such as Escherichia coli (three different types; for a review see [5]) [6, 71,, Chloroflexus uurantiacus [9] and several lactic acid bacteria [lo]. It has been shown for most of these enzymes that they contain pyridoxal phosphate (for a review see [l 11) bound to a specific lysine residue via a Schiff base [12]. The amino acid sequences around this lysine residue in E. coli, yeast, rat and man are conserved [I, 3, 131. During catalysis the electron-withdrawing prosthetic group forms a new Schiff base with the substrate whereby the removal of the proton from the a-carbon of the hydroxy amino acid is facilitated [14, 151.
The generation of transport vesicles at the endoplasmic reticulum (ER) depends on cytosolic proteins, which, in the form of subcomplexes (Sec23p͞Sec24p; Sec13p͞ Sec31p) are recruited to the ER membrane by GTP-bound Sar1p and form the coat protein complex II (COPII). Using affinity chromatography and two-hybrid analyses, we found that the essential COPII component Sec24p, but not Sec23p, binds to the cis-Golgi syntaxin Sed5p.
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