The Role of CDC48 in the Retro-translocation of Non-ubiquitinated Toxin Substrates in Plant Cells

Richard S Marshall,Nicholas A Jolliffe,Aldo Ceriotti,Christopher J Snowden,J Michael Lord,Lorenzo Frigerio,Lynne M Roberts

doi:10.1074/jbc.m709316200

Richard S Marshall, Nicholas A Jolliffe + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m709316200

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Abstract

When the catalytic A subunits of the castor bean toxins ricin and Ricinus communis agglutinin (denoted as RTA and RCA A, respectively) are delivered into the endoplasmic reticulum (ER) of tobacco protoplasts, they become substrates for ER-associated protein degradation (ERAD). As such, these orphan polypeptides are retro-translocated to the cytosol, where a significant proportion of each protein is degraded by proteasomes. Here we begin to characterize the ERAD pathway in plant cells, showing that retro-translocation of these lysine-deficient glycoproteins requires the ATPase activity of cytosolic CDC48. Lysine polyubiquitination is not obligatory for this step. We also show that although RCA A is found in a mannose-untrimmed form prior to its retro-translocation, a significant proportion of newly synthesized RTA cycles via the Golgi and becomes modified by downstream glycosylation enzymes. Despite these differences, both proteins are similarly retro-translocated.

Highlights

As in mammalian and yeast cells, the plant cell endoplasmic reticulum (ER)3 is a major protein folding compartment
A pathway closely resembling ER-associated protein degradation (ERAD) operates in plant cells but, by comparison, relatively little is known about substrate recognition, extraction from the ER membrane, or the pathway(s) and machineries for degradation [13]
We have studied the effects of a dominant negative form of Arabidopsis CDC48 on the export of the orphan catalytic polypeptides of ricin and its close relative, Ricinus communis agglutinin, in tobacco protoplasts

Summary

EXPERIMENTAL PROCEDURES

Recombinant DNA—All DNA constructs were generated in the CaMV 35S-promoter-driven expression vectors pDHA (see Ref. 19, for toxin- and phaseolin-based constructs) and pamPAT-MCS (GenBankTM accession number AY436765 [16]) or pGreenII-0029 (see Ref. 20, for CDC48- or fluorescent proteinbased constructs). Protoplast Fractionation—Protoplast pellets (from 500,000 cells) were resuspended in 140 ␮l of 12% sucrose buffer (100 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM EDTA, 12% (w/w) sucrose, supplemented immediately before use with CompleteTM protease inhibitor mixture (Roche Applied Science)) and homogenized by pipetting 50 times with a Gilson-type micropipette through a 200-␮l tip. Protease Protection Assay—Protoplast pellets (from 500,000 cells) were homogenized in 12% sucrose buffer as described above, this time omitting protease inhibitors, and cell debris was removed by centrifugation at 500 ϫ g for 5 min at 4 °C. Preparation of Protein Extracts and Immunoprecipitation— Frozen samples were homogenized by adding 2 volumes of cold protoplast homogenization buffer (150 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1.5 mM EDTA, 1.5% (w/v) Triton X-100, supplemented immediately before use with CompleteTM protease inhibitor mixture). ER chaperones will not be induced in a proportion of the cells taken for analysis

Expression of Dominant Negative

RESULTS

Findings

DISCUSSION