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Vol. 10, Issue 8, 2519-2530, August 1999
Max-Delbrück-Laboratorium, D-50829 Köln, Germany
The split-Ubiquitin (split-Ub) technique was used to map the
molecular environment of a membrane protein in vivo. Cub,
the C-terminal half of Ub, was attached to Sec63p, and Nub,
the N-terminal half of Ub, was attached to a selection of differently
localized proteins of the yeast Saccharomyces
cerevisiae. The efficiency of the Nub and
Cub reassembly to the quasi-native Ub reflects the
proximity between Sec63-Cub and the Nub-labeled
proteins. By using a modified Ura3p as the reporter that is released
from Cub, the local concentration between
Sec63-Cub-RUra3p and the different
Nub-constructs could be translated into the growth rate of
yeast cells on media lacking uracil. We show that Sec63p interacts with
Sec62p and Sec61p in vivo. Ssh1p is more distant to Sec63p than its
close sequence homologue Sec61p. Employing Nub- and
Cub-labeled versions of Ste14p, an enzyme of the protein
isoprenylation pathway, we conclude that Ste14p is a membrane protein
of the ER. Using Sec63p as a reference, a gradient of local
concentrations of different t- and v-SNARES could be visualized in the
living cell. The RUra3p reporter should further allow the selection of
new binding partners of Sec63p and the selection of molecules or
cellular conditions that interfere with the binding between Sec63p and
one of its known partners.
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