Chapter 14. Specificity of Membrane Fusion Encoded in SNARE Proteins

Introduction

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 one another’s ability to become excited.) On fusion of these liposomes with those lacking the fluorescent lipids, the fluorescent lipids are diluted, and quenching is alleviated. Three sets of liposomes were 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.

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

Only specific v-SNARE and t-SNARE combinations result in fusion. In this case, v-SNARE 1 can induce fusion only with membranes containing t-SNAREs of the plasma membrane, whereas v-SNARE 2 induces fusion only with membranes containing those of the Golgi. v-SNARE 3 appears to be a bit more promiscuous, permitting fusion with membranes containing either plasma membrane or vacuolar t-SNAREs, though it induces more rapid fusion with those of the vacuole.

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

Because v-SNARE 1 induces fusion with the plasma membrane, this v-SNARE might be expected to be on vesicles that emerge from the trans-Golgi network. Alternatively, it might be present on endosomal membranes that cycle in and out of the plasma membrane. v-SNARE 2 fuses with the Golgi and thus might be expected to be on vesicles that mediate transport between the ER and Golgi or within the Golgi. Alternatively, v-SNARE 2 might be on vesicles moving retrogradely from the plasma membrane back to the Golgi. v-SNARE 3 might be on vesicles that move between the Golgi and the vacuole or between the vacuole and the plasma membrane.

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

In yeast, mutations in a specific v-SNARE gene would be useful for deter-mining the specific function of that SNARE. Temperature-sensitive mutations could be quite helpful, as incubation at the restrictive temperature may result in an increase in the number of unfused vesicles carrying cargo and these might be assessed to determine where in the secretory pathway these vesicles likely reside.

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

The data show that the cytoplasmic domain of v-SNARE 2 competitively interferes with the ability of liposomes containing v-SNARE 2 to fuse with liposomes containing Golgi t-SNARES. Competition occurs only if the cytoplasmic domain is mixed with the liposomes containing the Golgi t-SNARES and not if it is incubated with liposomes containing v-SNARE 2. These findings indicate that the cytoplasmic domain of v-SNARE 2 binds to the Golgi t-SNARES and thereby interferes with the t-SNARES’ ability to bind to intact v-SNARE 2. Yeast that overexpress the cytoplasmic domain of v-SNARE 2 would be predicted to exhibit a defect in the secretory pathway (similar to a dominant negative mutation in v-SNARE 2) at the fusion step in which this SNARE is needed.