Involvement of the CTerminal KDEL Sequence in Formation of KDELVesicles and in Efficient Vacuolar Transport

Although most plant papain-type proteases are transported to vacuoles or secreted along the general secretory pathway, KDEL-tailed proteases are packed into KDEL vesicles, which directly fuse with vacuoles through a Golgi apparatus-independent pathway. The most notable difference between normal papain-type protease and KDEL-tailed protease is the presence of a KDEL sequence at the C-terminus, suggesting that the KDEL-tail is involved in this

Fig. 2 Electron photographs showing the transport of SH-EP via KDEL-vesicles by bypassing the Golgi apparatus. A,B The anti-SH-EP antibody did not immunogold-label the Golgi apparatus, although the KDEL-vesicle was labeled. C Antibodies to SH-EP (10-nm particles) and an asparaginyl endopeptidase (15-nm particles) selectively labeled the KDEL-vesicle and the Golgi apparatus, respectively. D The anti-complex glycan antibody (15-nm particles) and anti-SH-EP antibodies (10-nm particles) specifically immunogold-labeled the Golgi-apparatus and KDEL-vesicle, respectively. E KDEL-vesicle immunogold-labeled with the anti-SH-EP antibody fused with a protein storage vacuole (PSV). Gold particles localized in the PSV as well as the KDEL-vesicle. F Ultrastructural photograph showing that a vesicle similar to the KDEL-vesicle fuses to PSV. Arrowheads indicate the region where the KDEL-vesicle merges with the PSV membrane. ER, endoplasmic reticu-lum; G, Golgi apparatus; KV, KDEL-vesicle; Mt, mitochondrion; PSV, protein storage vacuole. Bars, 200 nm

Fig. 2 Electron photographs showing the transport of SH-EP via KDEL-vesicles by bypassing the Golgi apparatus. A,B The anti-SH-EP antibody did not immunogold-label the Golgi apparatus, although the KDEL-vesicle was labeled. C Antibodies to SH-EP (10-nm particles) and an asparaginyl endopeptidase (15-nm particles) selectively labeled the KDEL-vesicle and the Golgi apparatus, respectively. D The anti-complex glycan antibody (15-nm particles) and anti-SH-EP antibodies (10-nm particles) specifically immunogold-labeled the Golgi-apparatus and KDEL-vesicle, respectively. E KDEL-vesicle immunogold-labeled with the anti-SH-EP antibody fused with a protein storage vacuole (PSV). Gold particles localized in the PSV as well as the KDEL-vesicle. F Ultrastructural photograph showing that a vesicle similar to the KDEL-vesicle fuses to PSV. Arrowheads indicate the region where the KDEL-vesicle merges with the PSV membrane. ER, endoplasmic reticu-lum; G, Golgi apparatus; KV, KDEL-vesicle; Mt, mitochondrion; PSV, protein storage vacuole. Bars, 200 nm unique vacuolar trafficking pathway. To address the possibility, SH-EP and its KDEL-deletion mutant (SH-EP AKDEL) were heterologously expressed in Ara-bidopsis and intracellular localizations of these proteins were monitored. In cells from stems, cotyledons, rosette leaves and flowers of transformed plants expressing SH-EP, the enzyme accumulated in vesicles with diameters between 200-700 nm (Fig. 3A,B; Okamoto et al. 2003). These vesicles possibly correspond to KDEL-vesicles, since their size and the accumulation of KDEL-tailed protease are characteristic of KDEL-vesicles in cotyledons of mung bean seedlings. In addition, KDEL-vesicles in transgenic plants appeared to fuse with vacuoles (Fig. 3A). In contrast to heterologous expression of intact SH-

Fig. 3 Heterologous expression of SH-EP (A,B,E) and SH-EP AKDEL (C,D,E) in Arabidopsis (A-D) and tobacco BY2 cells (E,F). A,B Electron micrographs showing the development of KDEL-vesicles in stem (C) and cotyledon cells (D) of transgenic Arabidopsis expressing SH-EP. A KDEL-vesicle fused with a vacuole (arrowheads in panel A). C,D Electron photographs showing secretion of SH-EP AKDEL from the cells of rosette leaves of transgenic Arabidopsis. Gold particles from the anti-SH-EP antibody were found at extracellular spaces and possible air spaces. E,F Intracellular localization of signal peptide (SP)-GFP-SHEP (E) or SP-GFP-SHEP AKDEL (F) in tobacco BY-2 cells. Small foci derived from GFP fused with SH-EP were observed in the cells of panel E, however such signal was not detected in the cells of panel F. Bars, 200 nm (A-D) and 50 |im (E,F)

Fig. 3 Heterologous expression of SH-EP (A,B,E) and SH-EP AKDEL (C,D,E) in Arabidopsis (A-D) and tobacco BY2 cells (E,F). A,B Electron micrographs showing the development of KDEL-vesicles in stem (C) and cotyledon cells (D) of transgenic Arabidopsis expressing SH-EP. A KDEL-vesicle fused with a vacuole (arrowheads in panel A). C,D Electron photographs showing secretion of SH-EP AKDEL from the cells of rosette leaves of transgenic Arabidopsis. Gold particles from the anti-SH-EP antibody were found at extracellular spaces and possible air spaces. E,F Intracellular localization of signal peptide (SP)-GFP-SHEP (E) or SP-GFP-SHEP AKDEL (F) in tobacco BY-2 cells. Small foci derived from GFP fused with SH-EP were observed in the cells of panel E, however such signal was not detected in the cells of panel F. Bars, 200 nm (A-D) and 50 |im (E,F)

EP, formation of KDEL-vesicle was not detected in cells of transformed plants expressing SH-EPAKDEL. Furthermore, SH-EPAKDEL was mainly secreted and localized at the extracellular spaces and possible air spaces (Fig. 3C,D), indicating that the mutated protease was not packed into KDEL-vesicle but was secreted. The KDEL-tail of the protease will be essential for formation of KDEL-vesicles and subsequent vacuolar transport via the vesicles.

Additional confirmation of involvement of the KDEL-tail in formation of KDEL-vesicles was from GFP-labeling of the vesicles by heterologous expression of GFP-fused SH-EP or SH-EPAKDEL in tobacco BY-2 cells. When signal peptide (SP)-GFP-SHEP was expressed, strong fluorescence was detected in small vesicles (Fig. 3E), and the diameter of the GFP-labeled small vesicles appeared to be similar to that of KDEL-vesicles, which were detected in cells of transformed Arabidopsis expressing SH-EP (Fig. 3A,B). In the case of SP-GFP-SHEPAKDEL expression in tobacco cells, such a small vesicle was not observed, suggesting that deletion of the KDEL-tail from SH-EP resulted in loss of formation of the KDEL vesicle.

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