Abstract
SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) are cytoplasmically oriented membrane proteins that reside on vesicular carriers (v-SNARE) and target organelles (t-SNARE). The pairing of a stage-specific v-SNARE with its cognate t-SNARE may mediate the specificity of membrane traffic. In the yeast Saccharomyces cerevisiae transport between the endoplasmic reticulum and Golgi complex employs two v-SNAREs, Bos1p and Sec22p, each containing a domain that is related to the neuronal v-SNARE synaptobrevin. Sed5p, which is homologous to syntaxin, is the t-SNARE that functions at this stage of the secretory pathway. Here we report that regions of Bos1p and Sec22p, which are homologous to synaptobrevin, bind to the syntaxin-like domain of Sed5p. Furthermore, we demonstrate that efficient v-SNARE/t-SNARE interactions require the participation of both v-SNAREs, indicating that, unlike post-Golgi membrane traffic, the active form of the endoplasmic reticulum to Golgi v-SNARE is a heteromeric complex.
Highlights
The secretory pathway is composed of several distinct membrane-bound compartments, each with a unique set of proteins
Bet1p, which contains a domain that is related to SNAP-25, resides primarily on the endoplasmic reticulum (ER) instead of the target membrane (5, 6)
The vesicle contains a specific membrane protein (v-SNARE) Contains More Than One Subunit—Cells disrupted for the SEC22 gene display no growth defect at 30 °C
Summary
Yeast Strains and Genetic Techniques—Yeast strains used in this study were: SFNY26-6A (MAT␣, his4 – 619), ANY112 (MATa, bet[1], ura3–52), SFNY411 (MAT␣, sec[22–3], ura[3–52], ade[2– 801], leu2-⌬98), SFNY412 (MATa, bos[1], ura[3–52], leu2–3, 112), SFNY357 (MAT␣, ura[3–52], leu[2,3, 112], SEC22::URA3), SFNY358 (MATa, ura[3–52], leu2–3, 112), SFNY82 (Mata/␣, leu2–3, 112/leu[2,3, 112], ura3–52/ura[3– 52], BET1::LEU2), SFNY571 (MAT␣, sed[], ura[3–52], leu[2,3, 112], his3⌬200, trp1-⌬901, lys[2– 801], suc2-⌬9), and NY426 (MATa, ura[3–52], sec[22–3]). DNA Constructions—The cytoplasmic domains of Bet1p, Bos1p, and Sec22p were amplified by polymerase chain reaction using the appropriate primers as described before (6). In Vitro Binding Assays and Quantitation of Bound Bos1p and Sec22p—In vitro binding assays were performed by incubating varying amounts of His6-tagged proteins with 1 M GST fusion protein (immobilized on beads) in a 100-l reaction containing binding buffer (see above) and 0.5% Triton X-100. Western blot analysis was performed with anti-Bos1p (1:1,500 dilution) or anti-Sec22p (1: 1,250 dilution) antiserum using 125I-protein A. To determine the amount of His6-tagged fusion protein bound to the beads, samples were compared with standards of the appropriate recombinant protein (either His6-Bos1p or His6-Sec22p) and, following immunodetection, exposed to a PhosphorImaging plate and scanned onto a PhosphorImager. The blots were exposed to a PhosphorImaging plate, scanned onto a PhosphorImager, and analyzed with Molecular Dynamics Image Quant software (version 3.15)
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