Detection of transient in vivo interactions between substrate and transporter during protein translocation into the endoplasmic reticulum

The split-ubiquitin technique was used to detect transient protein interact ions in living cells. N-ub, the N-terminal half of ubiquitin (Ub), was fuse d to Sec62p, a component of the protein translocation machinery in the endo plasmic reticulum of Saccharomyces cerevisiae. C-ub, the C-terminal half of Ub, was fused to the C terminus of a signal sequence. The reconstitution o f a quasi-native Ub structure from the two halves of Ub, and the resulting cleavage by Ub-specific proteases at the C terminus of C-ub, serve as a gau ge of proximity between the two test proteins linked to N-ub and C-ub. Usin g this assay, we show that Sec62p is spatially close to the signal sequence of the prepro-alpha-factor in vivo. This proximity is confined to the nasc ent polypeptide chain immediately following the signal sequence. In additio n, the extent of proximity depends on the nature of the signal sequence. C- ub fusions that bore the signal sequence of invertase resulted in a much lo wer Ub reconstitution with N-ub-Sec62p than otherwise identical test protei ns bearing the signal sequence of prepro-alpha-factor. An inactive derivati ve of Sec62p failed to interact with signal sequences in this assay. These in vivo findings are consistent with Sec62p being part of a signal sequence -binding complex.