Saturation of the Endoplasmic Reticulum Retention Machinery Reveals Anterograde Bulk Flow

Abstract

We have studied the possible mechanisms of endoplasmic reticulum (ER) export and retention by using natural residents of the plant ER. Under normal physiological 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 treatment 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 retention mechanism is possible. Overexpression of calreticulin led to 100-fold higher levels in dilated globular ER cisternae as well as dilated nuclear envelopes and partial secretion of both BiP and calreticulin. This result shows that both molecules are competent for ER export and supports the concept that proteins are secreted by default. This result also supports previous 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 significant amounts of complex glycans, in contrast to secreted calreticulin. This result 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. INTRODUCTIONResident chaperones and folding enzymes, termed reticuloplasmins, constitute the majority of proteins in the lumen of the endoplasmic reticulum (ER) in most cell types. In contrast, newly synthesized secretory and vacuolar proteins usually are in the minority. The mechanism by which the cell ensures efficient export of secretory proteins to the Golgi apparatus while maintaining high levels of soluble reticuloplasmins in the ER lumen has been the subject of intense investigation during the past 15 years (Pelham, 1995; Hong, 1998).The model by which soluble proteins devoid of sorting information exit the cell by default (Denecke et al., 1990; Hunt and Chrispeels, 1991; reviewed in Vitale and Denecke, 1999) implies that all molecules can diffuse freely into and out of nascent anterograde transport vesicles formed by the ER. Therefore, it has been assumed that ER resident proteins, such as the binding protein (BiP), can escape from the ER by diffusion into anterograde transport vesicles. A conserved C-terminal motif (mainly HDEL and KDEL in plants; reviewed in Vitale et al., 1993), which is recognized in the Golgi apparatus, is responsible for the retrieval of these proteins by the transmembrane receptor ERD2 (for ER retention defective 2; Lewis and Pelham, 1992). This recycling model is required to explain how a relatively small number of receptors could mediate retention of a large number of ligands wi...

Expression and localization of calreticulin in tobacco anthers and pollen tubes

“…Its apparent molecular weight (60 kDa) falls within the usual mobility range (50-60 kDa) shown by calreticulins run on SDS-PAGE and corresponds to the M r detected for calreticulin isolated from tobacco germinating seeds (Denecke et al 1995). Since tobacco calreticulin carries two consensus sites for N-linked glycosylation (Denecke et al 1995), the observed size difference between spinach (54 and 56 kDa) and tobacco (60 kDa) calreticulins may be partly attributed to the presence of two high-mannose glycan chains on the tobacco calreticulin (Crofts et al 1999).…”

“…The localization of calreticulin in the Golgi apparatus, including Golgi-coated vesicles and, at a lower abundance, in the ER that we found in tobacco pollen tubes may reflect Golgi-ER protein recycling if we take into account that tobacco calreticulin also carries two glycan chains of the typical ER high-mannose form (Crofts et al 1999).…”

Proteomic analysis of the Arabidopsis thaliana cell wall

“…The signal peptide does not have a consensus amino acid sequence, but is characterised by three domains; the positively charged n-region on the N-terminus and a central hydrophobic h-region followed by a polar c-region containing the cleavage site. Second, classical cell wall proteins do not possess the C-terminal KDEL or HDEL tetrapeptide, the canonical ER retention motif recognised by the mechanism that serves to prevent secretion of enzymes resident in the lumen of this organelle [32,33].…”

Plant Endoplasmic Reticulum

“…For example, cells with a high secretory load can develop a more cisternal ER, indicating that increasing the internal dimensions of the ER increases its capacity for protein synthesis (Figure 2; Crofts et al, 1999;Stephenson and Hawes, 1986;Ridge et al, 1999). In addition, changes in ER morphology to a more cisternal form around sites of pathogen invasion and wounding have been documented, and defects in proteins that control ER cisternalisation (RTNs) affect the plant's susceptibility to invasion (Hardham et al, 2008;Takemoto et al, 2003;Hwang and Gelvin, 2004;Lee et al, 2012).…”

“…Often, the ER has proved to be more benign to heterologous proteins or even proteins normally localised in the vacuoles, leading to increased protein yields in transgenic plants. The ER appears to have an enormous potential for morphological change, as exemplified by the globular ER of considerable size found in calreticulin-overproducing tobacco plants (Crofts et al, 1999) that were otherwise growing normally. The growing knowledge of the processes associated with protein synthesis, storage, ER export and recycling from the Golgi, and the potential role(s) of ER 'shape' and dynamics in these processes now open up the prospect of engineering the secretory pathway to optimise heterologous protein production.…”