Figure 7 Get3b interacts with the thylakoid membrane proteins
Alb3 and Alb4
(A) In vitro pull-down assays were performed with recombinant
GST-tagged mature Get3b (GST-Get3b) and the His-tagged C-terminus of
Alb3 (Alb3C-His) or Alb4 (Alb4C-His). Samples containing GST and the
C-termini, or the C-termini alone served as negative controls.
Approximately 4% of the load samples were applied to SDS-PAGE analysis
and stained with Coomassie. (B) 10% of the eluates of the
pull-down assay were subjected to immunoblot analysis using antibodies
specific for Alb3, Alb4, or GST. The asterisks indicate truncated forms
of the GST-Get3b. The closed circle indicates a nonspecific reaction of
the anti-Alb4 antibody with GST. (C) Split-ubiquitin yeast
two-hybrid assay using mature Get3b and mature full-length Alb3. Alb3
was used as fusion with the C-terminal half of ubiquitin (Cub) at its
C-terminus (Alb3-Cub). Get3b was used as fusion with a modified
N-terminal half of ubiquitin (NubG) at its N-terminus (NubG-Get3b). The
unrelated endoplasmic reticulum membrane protein Alg5 fused to wild-type
Nub (Alg5-NubI) and NubG (Alg5-NubG) served as positive and negative
controls for the Cub-fusion construct, respectively. DSY-1 yeast
colonies were first plated on permissive medium (−LT, lacking Leu and
Trp) and on selective medium (−LTH, lacking Leu, Trp, and His).(D) Same assay as in (C) but using mature full-length Alb4 as
bait. (E) Split-ubiquitin interaction tests of SecY1-Cub with
NubG-Get3b and of Alg5-Cub with NubG-Get3b as negative control.