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doi: 10.1242/10.1242/jcs.00027

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Binding of Sly1 to Sed5 enhances formation of the yeast early Golgi SNARE complex

Yoichi Kosodo*, Yoichi Noda, Hiroyuki Adachi and Koji Yoda{ddagger}

Department of Biotechnology, the University of Tokyo, Yayoi, Bunkyo-Ku, Tokyo 113-8657, Japan
* Present address: Max-Planck-Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, D-01307, Dresden, Germany



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  		Fig. 6. Disassembly of the cis-SNARE complex by NSF/Sec18 and stimulation of trans-SNARE complex assembly by Sly1. The cell-free assay system was carried out with or without adding ATP or its unhydrolyzable analog AMPPNP. Yeast lysate and a recombinant protein were mixed as described in Fig. 4. The mixture was supplemented with 1 mM ATP (lanes 2 and 6) or 1 mM AMPPNP (lanes 3 and 7) or had nothing added (lanes 1 and 5). The reactions were started by shifting the temperature to 35°C and continued for up to 30 minutes. In the experiments shown in lanes 4 and 8, the mixture with exogenous Sly1/Sly1ts-Strep was incubated in the presence of 1 mM ATP at 35°C for 30 minutes. Then 1 mM AMPPNP was added and the incubation was continued for another 30 minutes before addition of Triton X-100. The bands of Sly1/Sly1ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads. *indicates the endogenous Sly1ts protein in lysate I and II.

 


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  		Fig. 1. Sly1ts protein has a lower binding affinity for Sed5 than Sly1 protein. (A) Recombinant genes encoding myc-tagged Sly1, Sly1-20 and Sly1ts on CEN plasmids under its native promoter were expressed in the sly1ts strain. Lysates of the sly1ts strains containing vector, Sly1-6myc, Sly1-20-6myc or Sly1ts-6myc were prepared with glass beads and 1% Triton X-100, and immunoprecipitation was performed using an anti-myc monoclonal IgG and equal starting amounts of lysate. The Sly1-6myc/Sly1-20-6myc/Sly1ts-6myc protein and Sed5 protein immunoprecipitated by anti-myc antibody were detected by western blotting. The bands of Sly1-6myc/Sly1-20-6myc/Sly1ts-6myc and Sed5 are indicated. (B) MBP-fusion proteins of Sed5{Delta}TMD and its coiled-coil regions with a C-terminal myc tag were produced in E. coli and purified by amylose resin. GST-fusion Sly1/Sly1ts proteins were also produced and purified by glutathione-Sepharose 4B. Proteins were collected by anti-myc IgG and Protein A-Sepharose beads from the mixture of MBP-Sed5-myc (3 µg) and GST-Sly1 (6 µg, lane 1); MBP-Sed5-myc and GST-Sly1ts (6 µg, lane 2); MBP-Sed5H1-myc (3 µg) and GST-Sly1 (lane 3); MBP-Sed5H1-myc and GST-Sly1ts (lane 4); MBP-Sed5H3-myc (3 µg) and GST-Sly1 (lane 5); MBP-Sed5H3-myc and GST-Sly1ts (lane 6). Note that equal amounts of GST-Sly1 and GST-Sly1ts proteins were used for the binding assay (6 µg). The bands of MBP-Sed5-myc, MBP-Sed5H1-myc, MBP-Sed5H3-myc and GST-Sly1/Sly1ts are indicated by arrowheads on the right. Positions of molecular mass markers are shown on the left in kDa.

 


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  		Fig. 2. Sly1ts protein localizes on the membrane as does the wild-type Sly1 protein. The C-terminally myc-tagged Sly1, Sly1-20 and Sly1ts proteins were produced in the sly1ts strain from CEN plasmids. Cells containing Sly1-6myc (lanes 1-3), Sly1-20-6myc (lanes 4-6) and Sly1ts-6myc (lanes 7-9) were lysed by vortexing with glass beads and were subjected to centrifugation at 1,000 g for 5 minutes to remove unbroken cells and debris. The supernatant was fractionated by differential centrifugation at 10,000 g for 10 minutes and 100,000 g for 1 hour at 4°C. Aliquots of the fractions were subjected to SDS-PAGE and western blot analysis with antibodies against the localization marker proteins shown to the left.

 


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  		Fig. 3. Sly1 is included in the Bet1-Sed5 v-t-SNARE complex in vivo. The SLY1 gene of the wild-type strain W303 was substituted with an epitope-tagged SLY1-6myc. Cells incubated in YEPD medium at 30°C were lysed by vortexing with glass beads on ice. After adding 1% Triton X-100, the lysate was divided into three parts, and each was immunoprecipitated at 4°C by a monoclonal anti-myc antibody, by an affinity-purified polyclonal anti-Sed5 antibody or by the preimmune rabbit serum from which the anti-Sed5 antibody had arisen. Anti-myc, anti-Sed5 and anti-Bet1 antibodies were used to detect Sly1-6myc, Sed5 and Bet1 proteins in the immunoprecipitates (lanes 1-3) and one fifth of their supernatants (lanes 4-6). One fifth of starting lysate is shown in lane 7.

 


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  		Fig. 4. Functional Sly1 protein promotes or stabilizes the Bet1-Sed5 complex in vitro. The sly1ts strain was transformed with a recombinant 6myc-SED5 gene under the SED5 promoter on a CEN plasmid (I) or with a 3HA-BET1 gene under the GAL1 promoter on a 2µ plasmid (II). Preparation of the yeast lysates and recombinant proteins are described in Materials and Methods. The reaction time was fixed at 20 minutes, but the reaction temperature was 0°C (lanes 1 and 2), 15°C (lanes 3 and 4), 25°C (lanes 5 and 6) or 35°C (lanes 7 and 8). At the end of the reaction, the mixture was diluted and solubilized with Triton X-100 and subjected to immunoprecipitation using an anti-myc antibody. Western blotting was done to detect 6myc-Sed5, 3HA-Bet1 and Sly1/Sly1ts-Strep and endogenous Sly1ts protein in lysate I and II. The bands of Sly1/Sly1ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads. *indicates the endogenous Sly1ts protein in lysate I and II.

 


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  		Fig. 5. Time course of Bet1-Sed5 complex formation with Sly1 or Sly1ts. (A) The cell-free assay system was carried out at the various reaction times indicated. Yeast lysate and a recombinant protein were mixed as described in Fig. 4. The reactions were started by shifting the temperature to 35°C and were continued for up to 20 minutes. The bands of Sly1/Sly1ts-Strep, 6myc-Sed5 and 3HA-Bet1 are indicated by arrowheads at the right. *indicates the endogenous Sly1ts protein in lysate I and II. (B) Reactions were incubated for 20 minute at 0°C or 35°C, as in A, before the chemiluminescent signal of 3HA-Bet1 immunoprecipitated with 6myc-Sed5 was quantified by a luminoimage analyzer. The signal from the reaction at 0 °C with Sly1 was considered to be a control value. Signals from other reactions were counted and divided by the control value. The control value was subtracted from other values, and increased values of each reactions are indicated in the graph. The average of three experiments and standard error are indicated.

 

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