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. 2006 Sep;18(9):2275–2293. doi: 10.1105/tpc.105.040279

Figure 6.

Figure 6.

Specific Inhibition of Vacuolar Transport by SYP21 Overexpression.

(A) Transient expression experiment (16 h incubation) with tobacco leaf protoplasts electroporated with plasmids encoding the vacuolar cargo molecule GFP-spo and the PVC marker YFP-BP80. Shown is an image of the cell cortex excluding the vacuolar lumen from the pinhole to facilitate monitoring of the intermediate organelles of the secretory pathway (ER, Golgi, and PVC) contained within the cytosol. Signals from the two distinct fluorescent molecules are shown individually (in green and red, respectively) or as a merge. Images in bright field (differential interference contrast [DIC]) are shown at the right. Note the reticular patterns for the vacuolar cargo that is in transit through the ER. Bar = 5 μm.

(B) Similar experiment to (A) except that YFP-BP80 was replaced by YFP-SYP21. In contrast with (A), GFP-spo was enriched in well-defined punctate structures well above the background level in the reticular structures and perfectly colocalized with the punctate signals by YFP-SYP21. Bar = 5 μm.

(C) Transient expression experiment (24-h incubation) with tobacco leaf protoplasts electroporated with plasmids encoding the membrane-spanning protein YFP-SYP22. Shown is a cross section through a protoplast to allow visualization of the tonoplast. The control panel shows that no YFP signals bleed through to the GFP channel. The merge with DIC illustrates how the tonoplast meanders on the inside of the chloroplasts, which are found in between the tonoplast and the plasma membrane. Bar = 5 μm.

(D) Transient expression experiment as in (C) except that GFP-SYP21 was cotransfected with YFP-SYP22. Notice that under these conditions, YFP-SYP22 is colocalized in punctate structures together with GFP-SYP21, some of which are particularly enlarged (arrows). Shown is a cortex view of the protoplast. Bar = 5 μm.

(E) Transient expression experiment (24-h incubation) with tobacco leaf protoplasts transfected with plasmids encoding GFP-SYP21 and a YFP-tagged form of the plasma membrane syntaxin SYP121. Shown is a cross section of the protoplast to better visualize the plasma membrane. GFP-SYP21 overexpression does not influence the targeting of YFP-SYP121 to the plasma membrane. Bar = 5 μm.