In Vitro Translation and Translocat

In Vitro Translation and Translocation Next, we tested whether the observed mutations influenced the first steps of translation by using a cell-free in vitro translation and translocation system. In the absence of canine pancreatic microsomes, both wild-typeand mutant cRNA (exon 1a to exon 6) were translated to GABRB3 proteins of the same size, namely 30 kDa (Figure 7,wild-type lane S, supernatant protein). Such proteins corresponded to the preproteins of the b3 subunit before cleavage and contain the signal peptides and the unglycosylated GABRB3 protein. Canine pancreatic microsomal membranes (1.8 ml) (Promega, Madison, WI) were then added directly to each reaction medium for translocation experiments. After one hour of centrifugation, pellets were separated from supernatant. The supernatant fluid of all samples did not contain GABRB3 protein (not shown). The pellets, on the other hand, contained the GABRB3 protein that had been translocated into the microsome (Figure 7—see translocated proteins represented as ‘‘P’’). Translocation in the presence of 1.8 ml of canine pancreatic microsomes precipitated all beta 3 subunit protein into the pellet.This implied that all signal peptides were oriented toward the membrane and that all GABRB3 protein was translocated when incubating with 1.8 ml of canine pancreatic microsomes.