LETTER TO THE EDITOR

Reply: Glycobiology and the Plant Cell—A World of Information

Releasing appropriate information to members of the scientific community as well as re-emphasizing that glycobiology-related events are information-based processes call for our detailed answer to the letter by Pimpl and Denecke.

Indeed, the rapidly increasing amount of information now available on N-glycan maturation permits quick progress in the characterization of mechanisms that govern protein targeting along the plant secretory pathway. Our approach is based on carrot cell wall invertase (Inv) as a reporter glycoprotein fused with an endoplasmic reticulum (ER) retention signal (HDEL), a purification (His) tag, and an immunodetection (Flag) tag. This InvFlagHisHDEL was used as bait to collect information on N-glycan structures when a protein is retained in the ER exclusively on the basis of the HDEL-dependent recycling machinery. Actually, a major hurdle in this project was the selection of our reporter glycoprotein that, according to our specifications, had to be extracellular and stable, and that had to undergo N-glycan maturation during its transport through the Golgi apparatus. We also needed to prevent contamination of the ER resident reporter protein fused with HDEL by secreted forms of the same reporter that would escape the ER retention machinery. The carrot cell wall invertase appeared to be a perfect model, particularly because of the established cleavage of its C-terminal propeptide that naturally occurs during transport to the cell wall. Accordingly, we fused our immunopurification and immunodetection tags in a C-terminal position from this cleavable propeptide so that the minor amounts (<10%) of InvFlagHisHDEL that would escape the ER retention machinery to be secreted would have lost their tags and could not contaminate the products immunopurified from a microsomal fraction with antibodies specific for the Flag epitope. In a pulse–chase experiment, we have shown that the whole InvFlagHisHDEL retained in the ER progressively acquires EndoH and PNGase F resistance. According to what we currently know about the specificity of these endoglycosidases, this result indicates that InvFlagHisHDEL N-glycans mature and, notably, are –1,3-fucosylated during the signal-mediated recycling of the reporter from the Golgi apparatus (Pagny et al. 2000 ). Hence, retrieval of the whole ER resident invertase occurs down to the medial and trans Golgi. Due to this precise strategy and a glycan analysis of this "artificial reticuloplasmin," we have obtained strong evidence for a very active retrograde pathway between the Golgi and ER in plants.

Pimpl and Denecke also comment on our analysis of the natural ER resident protein calreticulin, and they compare our results (Pagny et al. 2000 ) to theirs (Crofts et al. 1999 ). In Figure 1 of their paper, traces of truncated calreticulin deleted from its HDEL C-terminal motif (calreticulinHDEL) are barely detectable in the incubation medium of tobacco protoplasts. Anyone who has cultured tobacco protoplasts over an extended incubation time (24 hr in Crofts et al. 1999 ) has observed that many protoplasts burst during incubation and, consequently, release their intracellular content, including ER proteins, vacuolar proteases, and the like into the incubation medium. Needless to say, this protease-rich medium is the perfect environment for uncontrolled proteolytic maturation of intracellular proteins released by protoplasts that have burst open. Under these conditions, we find highly questionable the authors' claims that (1) trace amounts of calreticulinHDEL found in the protoplast medium have been secreted and (2) the lower molecular weight of calreticulinHDEL found in this medium indicates that glycan processing has taken place during its transport from the ER to the cell surface (Crofts et al. 1999 ). Even if one admitted that the trace amounts of both BiPHDEL and calreticulinHDEL found in the protoplast incubation media (Crofts et al. 1999 ) do represent a fraction that is secreted after HDEL deletion and that would normally recycle if the HDEL signal were present, these results would strongly reinforce our conclusions, namely that ER retention of these proteins relies primarily on mechanisms other than HDEL-mediated recycling.

Surprisingly, Pimpl and Denecke consider that our results and their results are similar, whereas both teams have reached opposite conclusions. The results are not similar. In one case, Crofts et al. 1999 , the calreticulin N-glycan structure is so roughly defined with a low specificity endoglycosidase, that one cannot determine whether calreticulin recycles from the Golgi back to the ER. In the other case, the wide variety of tools now used by plant glycobiologists, such as glycan-specific antibody probes, lectins, highly specific endoglycosidases, and mass spectrometry (Bardor et al. 1999 ), has helped to determine that calre-ticulin harbors Man8GlcNAc2 and Man9GlcNAc2 N-glycans. The latter are competent for modification by Golgi enzymes and, hence, their structure does not support a recycling of calreticulin through the Golgi, not even through the early cis Golgi compartment where -1,2-mannosidase I is localized (Nebenfuhr et al. 1999 ).

Our results and those of Pimpl and Denecke are not similar unless one considers that stably transformed suspension-cultured tobacco cells, which have been studied after several months of subculturing after transformation (Pagny et al. 2000 ), will provide the same type of information about the secretory pathway as do protoplasts overexpressing a reporter protein at such a high level that ER cisternae and nuclear envelopes are dilated (Crofts et al. 1999 ). What could one seriously conclude about the normal transit downstream of such a heavily constipated ER? At best, that protein secretion occurs via a default pathway (i.e., a conclusion that is strongly supported by results obtained during the last century [ Denecke et al. 1990 ; Hunt and Chrispeels 1991 ]).

As illustrated in Pagny et al. 2000 , plant glycobiologists have the tools necessary to demonstrate that calreticulin is mainly retained in the ER by mechanisms that rely primarily on signals other than the HDEL motif. The C-terminal extension of this plant reticuloplasmin likely remains a "security signal" that, at least under physiological conditions, very efficiently prevents the chaperone from being secreted, should it ever escape the ER. Our continued investigations will help us to characterize the mechanism(s) and signal(s) that strongly limit calreticulin diffusion in vesicles exported from the ER.

REFERENCES

Bardor, M., Faye, L., and Lerouge, P. (1999) Analysis of the N-glycosylation of recombinant glycoproteins produced in transgenic plants. Trends Plant Sci. 4:376-380[CrossRef][ISI][Medline].

Crofts, A.J., Leborgne-Castel, N., Hillmer, S., Robinson, D.G., Phillipson, B., Carlsson, L.E., Ashford, D.A., and Denecke, J. (1999) Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow. Plant Cell 11:2233-2247[Abstract/Free Full Text].

Denecke, J., Botterman, J., and Deblaere, R. (1990) Protein secretion in plant cells can occur via a default pathway. Plant Cell 2:51-59[Abstract/Free Full Text].

Hunt, D.C., and Chrispeels, M.J. (1991) The signal peptide of a vacuolar protein is necessary and sufficient for the efficient secretion of a cytosolic protein. Plant Physiol. 96:18-25[Abstract/Free Full Text].

Nebenführ, A., Gallagher, L.A., Dunahay, T.G., Frohlick, J.A., Mazurkiewicz, A.M., Meehl, J.B., and Staehelin, L.A. (1999) Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system. Plant Physiol. 121:1127-1141[Abstract/Free Full Text].

Pagny, S., Cabanes-Macheteau, M., Gillikin, J.W., Leborgne-Castel, N., Lerouge, P., Boston, R.S., Faye, L., and Gomord, V (2000) Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention. Plant Cell 12:739-755[Abstract/Free Full Text].

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