Membrane-Bound KKRY–GFP Colocalizes with the ER Marker Protein BiP in Arabidopsis and Tobacco BY-2 Transformants.Arabidopsis and tobacco BY-2 transformants expressing the KKRY–GFP fusion were immunolabeled after aldehyde fixation with mouse monoclonal GFP–s... Membrane-Bound KKRY–GFP Colocalizes with the ER Marker Protein BiP in Arabidopsis and Tobacco BY-2 Transformants.Arabidopsis and tobacco BY-2 transformants expressing the KKRY–GFP fusion were immunolabeled after aldehyde fixation with mouse monoclonal GFP–specific antibodies, rabbit polyclonal BiP–specific antibodies, mouse monoclonal actin–specific antibodies, and rabbit polyclonal tubulin–specific antibodies, as indicated. Primary antibodies were detected with either mouse species–specific FITC or rabbit species–specific Alexa 568 conjugates. Confocal laser images are therefore presented in green pseudocolor for both the KKRY–GFP fusion and actin and in red pseudocolor for BiP and tubulin. Colocalization is indicated by yellow where the green and red colors are superimposed.(A) to (D) Arabidopsis cotyledon epidermis.(A) Anti-actin and anti-tubulin double labeling. A pair of guard cells and surrounding pavement cells show distinct distribution patterns for microtubules and actin bundles. Note the clean separation of FITC (green) and Alexa 568 (red) signals. Some residual GFP fluorescence is visible along with the actin label in the 5-μm-thick projection.(B) Anti-GFP and anti-BiP double labeling of a single pavement cell with a well-preserved cortical ER network in the upper 2.4 μm of the Z series. Autofluorescent cuticular material and chlorophyll are visible in the BiP and merged images.(C) Anti-GFP and anti-BiP double labeling of a guard cell with a cortical focus of 0 to 2 μm. Note the relatively weak residual GFP fluorescence in the adjacent guard cell. The fact that BiP localization is restricted to the lower guard cell indicates that only this cell was permeabilized.(D) Same region as shown in (C), with a midplane focus. Note the GFP- and BiP-specific fluorescence at the periphery of nuclei.(E) to (H) Tobacco BY-2 cells.(E) Anti-actin and anti-tubulin double labeling with clean separation of FITC and Alexa 568 signals in a 4-μm projection.(F) Anti-GFP and anti-BiP double labeling in a cortical region, ∼3 μm from the upper surface of the cell.(G) Anti-GFP and anti-BiP double labeling at the upper edge of the nucleus, ∼8 μm below the upper surface.(H) Anti-GFP and anti-BiP double labeling with a midplane focus ∼18 μm below the upper surface. Note the fluorescence at the nuclear envelope, transvacuolar strands, and cell cortex.Bar in (A) = 20 μm; bar in (B) = 20 μm for (B) to (D); bar in (E) = 20 μm; bar in (F) = 20 μm for (F) to (H). Mohammed Benghezal et al. Plant Cell 2000;12:1179-1201 ©2000 by American Society of Plant Biologists