Involvement of the Golgi apparatus in vacuolar transport of KDEL-tailed proteases has been observed in detail, since vacuolar proteins are generally transported along the secretory pathway via the Golgi apparatus, from which vacuolar and secretory proteins are separately sorted. Despite the immunogold-labeling of ER and KDEL-vesicles in cotyledon cells of mung bean seedlings with anti-SH-EP antibody, the Golgi apparatus was never labeled with this antibody (Fig. 2A,B). On the other hand, when an antibody to another vacuolar protease, an asparaginyl endopeptidase, was employed for immunogold labeling, the Golgi apparatus and vacuoles were labeled, but the KDEL-vesicle was not (Fig. 2C). This selective labeling of KDEL-vesicles and Golgi apparatus with two kinds of vacuolar proteases indicates that cotyledon cells use two sorting pathways to transport proteolytic enzymes from the ER to vacuoles, a Golgi-mediated route for asparaginyl endopeptidase and a KDEL-vesicle mediated route for SH-EP. Moreover, a complex glycan antibody did not label KDEL-vesicles, but it did label the Golgi apparatus (Fig. 2D), indicating that proteins from the Golgi apparatus do not contribute to the content or formation of the vesicles. These immuno-cytochemical studies have shown that KDEL-vesicles bypass the Golgi apparatus and directly fuse with protein storage vacuoles (Fig. 2E,F). Bypassing the Golgi apparatus by KDEL-vesicles will probably be consistent even when organelle/vesicle sizes are compared, since the diameter of KDEL-vesicles (200-500 nm) is probably too large for discharge of their content into the Golgi apparatus without extreme structural consequences to this organelle.
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