In order to examine the specificity of membrane fusion conferred by specific v-SNAREs and t-SNAREs, researchers reconstituted

Question:

In order to examine the specificity of membrane fusion conferred by specific v-SNAREs and t-SNAREs, researchers reconstituted liposomes (artificial lipid membranes) with specific t-SNARE complexes or with v-SNAREs (see McNew et al., 2000, Nature 407:153-159). To measure fusion, the v-SNARE liposomes also contained a fluorescent lipid at a relatively high concentration such that its fluorescence is quenched. (Quenching is reduced fluorescence relative to that expected. In this case, quenching occurs because the fluorescent lipids are too concentrated and interfere with each other's ability to become excited.) On fusion of these liposomes with those lacking the fluorescent lipid, the fluorescent lipids are diluted, and quenching is alleviated. Three sets of liposomes were prepared prepared using yeast t-SNARE complexes: those containing plasma membrane t-SNAREs, Golgi t-SNAREs, or vacuolar t-SNAREs. Each of these was mixed with fluorescent liposomes containing one of three different yeast v-SNAREs. The following data were obtained.

v-SNARE 2 v-SNARE 3 v-SNARE 1 ESNARE = plasma membrane ESNARE = Golgi tSNARE = vacuolar Time after mixing Fluorescence i

a. What can be deduced from these data about the specificity of membrane fusion events?

b. Where might you expect to find v-SNAREs 1, 2, and 3 in yeast?

c. What kind of experiment could be designed to determine where in the secretory pathway a given v-SNARE is required in vivo?

d. The cytoplasmic domain of v-SNARE 2 has been expressed and purified from E. coli. Various amounts of this domain are incubated either with the Golgi t-SNARE liposomes or with v-SNARE 2 liposomes. The liposomes are then washed free of unbound protein. The various liposomes are then mixed, as indicated below, and the fluorescence of each sample is measured 1 hour after mixing. How can the data be explained? What would you predict the outcome to be if yeast were to overexpress the cytoplasmic domain of v-SNARE 2?

= v-SNARE 2 liposomes incubated with the cytoplasmic domain of v- SNARE 2 and then mixed with Golgi t-SNARE liposomes =