Saturation of the endoplasmic reticulum retention machinery reveals anterograde bulk flow

We have studied the possible mechanisms of endoplasmic reticulum (ER) expor t and retention by using natural residents of the plant ER, Under normal ph ysiological conditions, calreticulin and the lumenal binding protein (BiP) are efficiently retained in the ER. When the ER retention signal is removed , truncated calreticulin is much more rapidly secreted than truncated BiP, Calreticulin carries two glycans of the typical ER high-mannose form. Both glycans are competent for Golgi-based modifications, as determined from tre atment with brefeldin A or based on the deletion of the ER retention motif, In contrast to BiP, calreticulin accumulation is strongly dependent on its retention signal, thereby allowing us to test whether saturation of the re tention mechanism is possible. Overexpression of calreticulin led to 100-fo ld higher levels in dilated globular ER cisternae as well as dilated nuclea r envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the con cept that proteins are secreted by default. This result also supports previ ous data suggesting that truncated BiP devoid of its retention motif can be retained in the ER by association with calreticulin. Moreover, even under these saturating conditions, cellular calreticulin did not carry significan t amounts of complex glycans, in contrast to secreted calreticulin. This re sult shows that calreticulin is rapidly secreted once complex glycans have been synthesized in the medial/trans Golgi apparatus and that the modified protein does not appear to recycle back to the ER.