ER quality control can lead to retrograde transport from the ER lumen to the cytosol and the nucleoplasm in plants

Abstract

SummaryQuality control in the secretory pathway is a fundamental step in preventing deleterious effects that may arise by the release of malfolded proteins into the cell or apoplast. Our aims were to visualise and analyse the disposal route followed by aberrant proteins within a plant cell in vivo using fluorescent protein technology. A green fluorescent protein (GFP) fusion was detected in the cytosol and the nucleoplasm in spite of the presence of an N-terminal secretory signal peptide. In contrast to secreted GFP, the fusion protein was retained in the cells where it was degraded slowly, albeit at a rate much higher than that of the endoplasmic reticulum (ER)-retained derivative GFP-HDEL. The fusion protein could not be stabilised by inhibitors of transport or the cytosolic proteasome. However, the protein is a strong lumenal binding protein (BiP) ligand. Complete signal peptide processing even after long-term expression in virus-infected leaves rules out the possibility that the documented accumulation in the cytosol and nucleoplasm is because of the bypassing of the translocation pores. The data are consistent with the hypothesis that the fusion protein is disposed off from the ER via a retrograde translocation back to the cytosol. Moreover, accumulation in the nucleoplasm was shown to be microtubule dependent unlike the well-documented diffusion of cytosolically expressed GFP into the nucleoplasm. The apparent active transport of the GFP fusion into the nucleoplasm may indicate an as yet undiscovered feature of the ER-associated degradation (ERAD) pathway and explain the insensitivity to degradation by proteasome inhibitors.

Advances in Fluorescent Protein-Based Imaging for the Analysis of Plant Endomembranes

“…In our lab, for example, we have initiated a large analysis of Arabidopsis EMS mutants stably expressing fluorescent markers of endomembranes to find novel factors that regulate the activity and integrity of organelles of the early plant secretory pathway. Using EMS mutagenesis of stable Arabidopsis plants expressing a secretory GFP retained in the ER (ssGFP-HDEL; Batoko et al, 2000;Brandizzi et al, 2003), followed by confocal microscopy screening, we have been able to identify mutants with obvious alterations of the ER pattern (Fig. 2).…”

Overexpression of theArabidopsisSyntaxin PEP12/SYP21 Inhibits Transport from the Prevacuolar Compartment to the Lytic Vacuole in Vivo

“…The underlined regions refer to engineered restriction sites for insertion in pLL4 (Brandizzi et al, 2003) between the CaMV35S promoter at the ClaI site overlapping with the start codon and the XbaI site overlapping with the stop codon preceding the 39-untranslated end of the nopaline synthase gene (39nos). This created the chimeric expression plasmid pOF19 to produce untagged natural wild-type SYP21 in plant cells.…”

“…To engineer SYP21 and SYP21DTM fusions to the GFP, an EcoRI-BglII fragment carrying the CaMV35S promoter followed by the GFP coding region was cut out of the cytosolic GFP expression plasmid cGFP described previously (Brandizzi et al, 2003) and ligated together with an annealed BglII-ClaI linker into either plasmid pOF19 for fusion to SYP21 or pOF31 for fusion to SYP21DTM. The linker was created by annealing the sense (59-GATCTCAGCAGGTGGAGCAT-39) and antisense (59-CGATG-CTCCACCTGCTGA-39) oligos, yielding a fragment encoding the symmetric linker peptide SerAlaGlyGlyAlaSer for optimal positioning and functional maintenance of the SYP21 function within the context of a GFP fusion.…”

Zeolin. A New Recombinant Storage Protein Constructed Using Maize γ-Zein and Bean Phaseolin

“…Interactions between wild-type zeins and BiP have not been studied, but BiP interacts with rice (Oryza sativa) prolamins during their cotranslational translocation into the ER lumen and has been detected at the periphery of rice protein bodies, from which it can be released by ATP treatment in vitro, suggesting long-lasting interactions (Li et al, 1993;Muench et al, 1997). BiP has also high affinity for severely defective proteins, which often extensively interact with BiP before being eventually degraded by ER quality control (for plant examples, see Pedrazzini et al, 1997;Brandizzi et al, 2003;Nuttall et al, 2003). Zeolin is very stable, and its interactions with BiP are extensive.…”