The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta -- Data Supplement - Supplemental Data -- Proceedings of the National Academy of Sciences

Supplementary material for Giranton et al., Proc. Natl. Acad. Sci. USA, 10.1073/pnas.070025097

Fig. 6.
Localization and topology of recombinant SRK in insect cells. (A) Subcellular localization. Sf21 cells expressing SRK₃HA were fixed with or without a permeabilization step, and immunofluorescence was performed using mAbs 85-36-71 or 12CA5 as indicated on the left. For each assay, a negative control (on the right) was performed as described in the supplementary Materials and Methods. Nonpermeabilized cells treated with mAb 85-36-71 exibited very low staining, and the antibody appeared to specifically label small vesicles. However, after permeabilization, cells were strongly stained and exhibited small highly fluorescent areas that apparently corresponded to local concentrations of recombinant proteins. These results suggested that only a small fraction of recombinant SRK₃ was addressed to the plasmalemma, the majority being retained under the cell surface. The use of mAb 12CA5 gave a more diffuse staining, suggesting that the plasmalemma of Sf21 cells is permeable to some extent to antibodies, even in the absence of Triton X-100. Nevertheless, the possibility that some recombinant SRK₃ molecules exposed their C termini at the outer cell surface cannot be ruled out. A very low level of fluorescence was detected in the negative controls performed, indicating that immunostaining was specific. Arrowheads indicate highly fluorescent spots. (Bar = 10 mm.) (B) Recombinant SRK is anchored in the membrane. Proteins were extracted from Sf21 cells expressing SRK₃HA in the presence (+) or absence (–) of 1% (mass/vol) Triton X-100, separated by SDS/PAGE, and electroblotted. SRK protein was detected with mAb 85-36-71. (C) Topology of recombinant SRK protein in insect cell microsomes. Microsomes, prepared from cells expressing SRK₃HA, were incubated in the presence (+) or absence (–) of trypsin and with various concentrations of Triton X-100 [0, 0.1, and 0.25 for 0, 0.1, and 0.25% (mass/vol), respectively]. Proteins were separated by SDS/PAGE and electroblotted, and immunodetections were carried out with mAb 85-36-71 and anti-SRK₃C-dom as indicated at the bottom. The sensitivity of SRK to tryptic digestion indicated that recombinant SRK spanned the membrane and was orientated with its S-domain in the lumen of the microsomal vesicles. Arrowhead indicates the recombinant SRK.