LETTER TO THE EDITOR

ER Retention of Soluble Proteins: Retrieval, Retention, or Both?

Pagny et al. 2000 have recently published an article that touches on a very controversial and important process in the secretory pathway, the export of soluble proteins from the endoplasmic reticulum (ER). The results that are reported confirm data reported previously (Pedrazzini et al. 1997 ; Crofts et al. 1999 ; Frigerio et al. 1999 ); however, the authors reach an opposite conclusion. It therefore appears important and timely to initiate a discussion about this topic.

Without any doubt, the issue of ER export is far from settled and discussions have been ongoing for almost a decade (Armstrong 1995 ). Two models are currently competing for general acceptance. One model describes ER export as a non-selective diffusion into anterograde ER-derived transport vesicles (bulk-flow). The second model postulates that proteins are actively selected and enriched during ER export (active transport). The bulk-flow model is supported by experiments in vivo using mammalian cells (Wiedman et al., 1984, 1987) and plant cells (Denecke et al. 1990 ; Hunt and Chrispeels 1991 ). The active transport model arose from studies using in vitro systems; the strongest evidence emerging from the fact that purified ER-derived COPII vesicles were enriched for yeast -factor, a secretory cargo molecule, whereas the ER resident chaperone BiP was absent in these vesicles (Barlowe et al. 1994 ). Because of the strong evidence for both models, the possibility that bulk-flow and active selection operate in the same transport system, perhaps through different types of transport vesicles, should not be excluded (Vitale and Denecke 1999 ).

Pagny and co-workers have now shown that the ER resident protein calreticulin does not carry complex glycans even under conditions of ER stress in the floury 2 mutant of maize. This confirms results obtained with ER- retained assembly defective phaseolin (Pedrazzini et al. 1997 ), KDEL-tagged phaseolin (Frigerio et al. 1999 ), as well as results on calreticulin under normal physiological conditions or during overproduction of this protein and subsequent saturation of the ER retention machinery (Crofts et al. 1999 ). It is not known whether assembly defective phaseolin is capable of leaving the ER or whether it is recycled from the Golgi via association with BiP, but either model must incorporate the fact that the protein does not acquire complex glycan modifications. However, the rapid export of phaseolin from the ER is well established (Frigerio et al. 1998 ), and ER export of calreticulin was achieved via overexpression (Crofts et al. 1999 ). In these cases, retrieval and subsequent accumulation of complex glycan containing forms in the endoplasmic reticulum could not be detected (Crofts et al. 1999 ; Frigerio et al. 1999 ). The conclusion from these reports is that retrieval of HDEL proteins that escape from the ER occurs from a Golgi compartment devoid of glycan-processing enzymes, such as the cis-most and the cis-Golgi cisternae.

There is good evidence that calreticulin leaves the ER frequently. First, deletion of the HDEL motif causes secretion, which demonstrates its dependence on the HDEL motif to remain in the cells (Crofts et al. 1999 ). The retrieval of HDEL proteins from the Golgi complex is well established in yeast and mammalian cells, and the functional complementation of AtERD2 in yeast certainly demonstrates that the plant HDEL receptor functions in the same way (Lee et al. 1993 ). Second, truncated calreticulin accumulates in transgenic plants to much lower levels than wild-type calreticulin when overexpressed using a strong viral promoter (Crofts et al. 1999 ). The discrepancy between truncated, and wild-type calreticulin was approximately a factor of 100 between the best overproducing plants. This is likely due to degradation of the truncated molecule in a post-ER compartment after ER export, thus under-representing the amount secreted in the absence of HDEL.

Pagny and co-workers claimed that calreticulin does not leave the ER and that HDEL-mediated retrieval of this ER resident is so minor that it would be undetectable via biochemical analysis. This claim is contradictory to our own interpretations and is deduced indirectly from findings with the HDEL-tagged secretory protein invertase (InvFlagHisHDEL). The authors argue that for this cargo molecule, HDEL-mediated retrieval occurs also from post-cis-Golgi cisternae, and that complex glycan containing invertase is frequently recycled back to the ER. In our opinion, the authors have not presented evidence to support their claim. Pulse–chase experiments were conducted and subsequent cellular extracts (the authors refer to "intracellular medium") were obtained through homogenizing entire cells and subsequent centrifugation of debris. The supernatant will contain Golgi vesicles and transport vesicles in addition to ER, as well as proteins trapped within the cell walls. InvFlagHisHDEL carrying complex glycans may therefore be in transit through the Golgi apparatus or simply in the cell walls. Also, it appears strange that microsomes contained undetectable amounts of secretory InvFlagHis and the authors refer in the text to "exclusive detection in the medium" (Figure 3, Pagny et al. 2000 ), whereas, "intracellular medium" contained easily detectable amounts in Figure 6 (Pagny et al. 2000 ). The two figures contradict each other, particularly because InvFlagHis and InvFlagHisHDEL in the "intracellular medium" are suddenly equal in abundance (Figure 6).

Until it has not been excluded by direct experimental analysis that InvFlagHisHDEL containing complex glycans are not derived from the cell walls in Figure 6, but in fact are localized in the ER, it is premature to suggest that recycling of complex modified glycoproteins back to the ER occurs at all. Such analysis could be done simply by repeating the pulse–chase experiment with washed protoplasts prepared from the BY2 cells. But since a protoplast extract would also contain Golgi membranes and transport vesicles, proof for recycling to the ER would be the purification of ER membranes and dem-onstration of a magnesium shift in sucrose gradients of complex modified InvFlagHisHDEL. This would show that complex InvFlagHisHDEL has reached the ER.

We argue that even if InvFlagHis-HDEL does indeed recycle from the transGolgi back to the ER, this does not mean that ER export of calreticulin is marginal just because it does not recycle from the transGolgi. Perhaps calreticulin is degraded when it reaches a post-cis-Golgi compartment whereas invertase is more stable? It is also possible that the HDEL motif of calreticulin is better presented in its natural context and thus confers more complete recycling from the cis-Golgi apparatus. Perhaps this is not the case for InvFlagHis-HDEL, causing escape to more distal regions of the Golgi, a suggestion that can also be derived from its partial secretion. It has already been shown that tagging with ER retention motifs is not always sufficient to obtain efficient retention. KDEL-tagged phytohemagglutinin was only partially retained in the ER and the nuclear envelope, the majority still reached the vacuoles (Herman et al. 1990 ). Yet a significant proportion of the recombinant protein was now endo-H sensitive, which would correspond well with the model in which retrieval of ER residents occurs mainly from the cis-Golgi apparatus.

Clearly, further work is needed to clarify the issue about ER export and retention of soluble proteins. This point is very important, as it also relates to the possible mechanism of quality control and ER retention of malfolded proteins. Do malfolded proteins leave the ER and recycle via association with ER chaperones such as BiP, or are they excluded from ER export? A constructive discussion and renewed research will certainly provide answers to these questions.

REFERENCES

Armstrong, J. (1995) Less bulk, more flow—Escape from the ER controversy. Trends Cell Biol. 5:149-150.

Barlowe, C., Orci, L., Yeung, T., Hosobuchi, M., Hamamoto, S., Salama, N., Rexach, M.F., Ravazzola, M., Amherdt, M., and Schekman, R. (1994) COPII: A membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum. Cell 77:895-907[CrossRef][ISI][Medline].

Crofts, A.J., LeborgneCastel, N., Hillmer, S., Robinson, D.G., Phillipson, B., Carlsson, L., Ashford, D.A., and Denecke, J. (1999) Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow. Plant Cell 11:2233-2248[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].

Frigerio, L., deVirgilio, M., Prada, A., Faoro, F., and Vitale, A. (1998) Sorting of phaseolin to the vacuole is saturable and requires a short C-terminal peptide. Plant Cell 10:1031-1042[Abstract/Free Full Text].

Frigerio, L., Pastres, A., Prada, A., and Vitale, A. (1999). KDEL acts before the Golgi-mediated formation of complex asparagine-linked glycans. In Annual Meeting of the American Society of Plant Physiologists, Baltimore. Abstract.

Herman, E.M., Tague, B.W., Hoffman, L.M., Kjemtrupp, S.E., and Chrispeels, M.J. (1990) Retention of phytohemagglutinin with carboxy terminal tetrapeptide KDEL in the nuclear envelope and the endoplasmic reticulum. Planta 182:305-312.

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].

Lee, H.I., Gal, S., Newman, T.C., and Raikhel, N.V. (1993) The Arabidopsis endoplasmic-reticulum retention receptor functions in yeast. Proc. Natl. Acad. Sci. USA 90:11433-11437[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 12:355-739.

Pedrazzini, E., Giovinazzo, G., Bielli, A., de Virgilio, M., Frigerio, L., Pesca, M., Faoro, F., Bollini, R., Ceriotti, A., and Vitale, A. (1997) Protein quality control along the route to the plant vacuole. Plant Cell 9:1869-1880[Abstract].

Vitale, A., and Denecke, J. (1999) The endoplasmic reticulum—Gateway of the secretory pathway. Plant Cell 11:615-628[Free Full Text].

Wiedmann, M., Huth, A., and Rapoport, T.A. (1984) Xenopus oocytes can secrete bacterial beta-lactamase. Nature 309:637-639[Medline].

Wieland, F.T., Gleason, M.L., Serafini, T.A., and Rothman, J.E. (1987) The rate of bulk flow from the endoplasmic-reticulum to the cell-surface. Cell 50:289-300[CrossRef][ISI][Medline].

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