Supplemental Material

This article contains the following supporting material:

• Figure 1 - Subcellular fractionation of lysates prepared from NY2547, NY2548 and NY2549 was performed as described in Methods section. Lysates were spun at 10,000 x g followed by a 100,000 x g spin. The resulting soluble (S) and pelletable (P) fractions were resolved on SDS-PAGE gel and different protein markers were detected by the appropriate antibodies. While wild type Sec2p is mostly found in S100 fraction, Sec2-59p and Sec2-78p redistribute to P100. Sec4p (Elkind et al., 2000) as well as the exocyst subunits Sec15p and Sec10p show normal fractionation pattern in sec2-59 and sec2-78 cells. The distribution of Sec22p ( a marker for ER and Golgi) and Pma1p (plasma membrane marker) was similar in all the examined strains.

• Figure 2 - An example of the distribution of Sec10p, Ypt32p, Sec4p and Trs33p in velocity glycerol gradients of lysates prepared from NY2546, NY2547 and NY2549. In these strains, all the markers examined behaved similarly. Ypt32p and Sec4p remained largely on top of the gradients while Sec10p co-sedimented with Sec15p. As exocyst is found in several fractions in this gradients, we examined the distribution of Trs33p (subunit of another large tethering complex, TRAPP) and found it to be distributed over multiple fractions as well.

• Figure 3 - Kex2p and invertase activities in sorbitol velocity gradient fractions. Top panel; Kex2p activity in wild type (NY2547; empty squares) and sec5-24 (NY 2646; closed squares) cells and invertase activity in sec5-24 (NY 2646; closed triangles) were measured as described in Methods section. Cells were grown overnight in YP medium containing 2% glucose. Wild type cells were lysed and fractionated as described in Methods section and Fig. 7D. To accumulate invertase enzyme, sec5-24 cells were first shifted to 37 0C in YP medium containing 0.2% glucose for 60 minutes and then lysed and fractionated as in Fig. 7D. The distribution of Sec4p across the gradient in sec5-24 strain was also examined (bottom panel).

• Figure 4 - Localization of the exocyst subunits in the wild type (NY2440, NY2441, NY2443 and NY2446) and sec2-59 (NY2638, NY2639, NY2640, NY2641) cells. Yeast were grown at 25 0C to log phase followed by a 60 minute shift to 37 0C. For GFP visualization, cells were harvested, fixed in methanol and acetone, washed with PBS and directly observed. Images were acquired as described in Methods section. Exocyst subunits known to depend on membrane traffic (Sec5-GFP, Sec8-GFP, Exo84-GFP and largely Exo70-GFP) are mislocalized in sec2-59 strain. In Sec3-GFP sec2-59 strain and partially in Exo70-GFP sec2-59 strain, proper localization to mother-daughter necks can be noticed. Attempts to introduce Sec6-GFP, Sec10-GFP and Sec15-GFP into sec2-59 background did not yield viable spores.

• Figure 5 - Sec2-59-GFP remains mislocalized in elp1D cells. Yeast (NY2147, NY2645) were grown at 25 0C in YPD to log phase and examined as described in Methods section.

• Figure 6 - Sec2p-Sec15p interaction is not significantly affected in elp1D cells. Following solubilization the microsomal pool (P100) of Sec2-GFP was immunoprecipitated with aGFP from wild type (NY2547, lane2) and elp1D (NY2642, lane3) cells. For a control, yeast strain expressing untagged Sec2p was used (NY2546, lane1). Likewise, P100 pool of Sec2-59-GFP and Sec2-78-GFP was immunoprecipitated with aGFP from wild type (NY2548, NY2549, lanes4 and 6) and elp1D (NY2643, NY2644, lanes5 and 7) cells. The experiment was preformed as described in Methods section. Sec15-13mycp in the immunoprecipitates was detected by amyc (top panel). All strains expressed similar amount of Sec15-13mycp (middle panel, 1% of the input is shown). The relative amounts of Sec2 proteins in P100 portions of yeast lysates is also shown (bottom panel). Efficiency of aGFP IP was 30 %.