Figure 1.
Alignment of CCI1-related proteins from land plants.
An alignment of Arabidopsis thaliana CCI1, CCI2 and related proteins from various plant species is shown. Conserved residues are shaded at 75%. Basic-rich regions are underlined. The top and bottom segments of CCI1 sequence correspond to the N and C-terminal half constructs used in the lipid-binding assays.
Figure 2.
CCI1 interactions with CLV signaling components in vitro and in transient expression.
A. Purified CLV1KD-GFP, GST, GST-CCI1 and GST-CCI2 proteins were mixed in various combinations, immunoprecipitated (IPd) with anti-GST antibodies, and the resulting immunoprecipitates were assayed on a protein gel blot probed with anti-GFP. The first lane shows CLV1KD-GFP input. B. Total membrane extracts from N. benthamiana leaves expressing CCI1-FLAG and full-length CLV1-GFP and BAM1-GFP IPd with anti-GFP antibodies and co-IP detected with anti-FLAG antibodies. Lanes 1, 2 and lanes 4, 5 are replicates. Note, CLV1-GFP did not express detectably in the lane 4 replicate, nor was there co-IP detected. Experiments represented by lanes 1–3 used an aliquot from the lane 7 expression of CCI1-FLAG alone (*). Co-IP was detected when both CCI1 and BAM1 were co-expressed in the same leaf (CCI1 co), but not when mixed post expression (CCI1 post). C. Total membrane extracts from N. benthamiana leaves expressing CCI1-FLAG and full-length BRI1-GFP, BAM2-GFP, CLV2-GFP and CRN-GFP IPd with anti-GFP antibodies and co-IP detected with anti-FLAG antibodies. Experiments represented by lanes 1–4 used an aliquot from the lane 9 expression of CCI1-FLAG alone (*). CoIP was detected when CCI1 and the receptors were co-expressed in the same leaf (CCI1 co), but not when mixed post expression (CCI1 post).
Figure 3.
CCI1 is plasma membrane localized.
A. Confocal image of CCI1-GFP transiently expressed in N. benthamiana leaves 48 hours after infiltration. Signal is detected at cell periphery. B. Two-phase membrane partitioning of CCI1-FLAG transiently expressed in N. benthamiana leaves. Endoplasmic reticulum marker BiP2 and plasma membrane marker PMA2 are used to mark the lower and upper phases, respectively.
Figure 4.
CLV pathway components partition to DRM/raft fractions.
A. Partitioning of CCI1-FLAG protein transiently expressed in N. benthamiana leaves into soluble membrane and DRM fractions. B. Partitioning of BAM1-FLAG, BAM2-FLAG and CLV2-MYC proteins in stable Arabidopsis transgenic lines into soluble membrane and DRM fractions is shown. C. Sucrose gradient sedimentation of solubilized membrane extracts of clatharin (as a control), CLV2-MYC and CRN-GFP from stable transgenic Arabidopsis meristem tissue. Lipid-associated proteins will float to the lighter fractions. D. Co-IP of transiently expressed BAM2-GFP and CCI1-FLAG was detected in both total membrane fraction (+) and membrane fraction after DRM depletion (–). Co-IP of transiently expressed CRN-GFP and CCI1-FLAG depended on the presence of DRMs.
Figure 5.
The N-terminal portion of CCI1 binds phospholipids.
Echelon membrane lipid strips and PIP strips probed with purified N-terminal GST tagged proteins at a concentration of 1 nM. The PH domain of the human FAPP protein which specifically binds PI(4)P was used as a positive control. The GST tag alone was used as a negative control. N-terminal CCI1 corresponds to the first 83 amino acids of the protein and C-terminal CCI1 corresponds to the remaining 70 (see Figure 1).