A Non-Classical Member of the Protein Disulfide Isomerase Family, PDI7 of Arabidopsis thaliana, Localizes to the cis-Golgi and Endoplasmic Reticulum Membranes

Abstract

Members of the protein disulfide isomerase (PDI)-C subfamily are chimeric proteins containing the thioredoxin (Trx) domain of PDIs, and the conserved N- and C-terminal Pfam domains of Erv41p/Erv46p-type cargo receptors. They are unique to plants and chromalveolates. The Arabidopsis genome encodes three PDI-C isoforms: PDI7, PDI12 and PDI13. Here we demonstrate that PDI7 is a 65 kDa integral membrane glycoprotein expressed throughout many Arabidopsis tissues. Using a PDI7-specific antibody, we show through immunoelectron microscopy that PDI7 localizes to the endoplasmic reticulum (ER) and Golgi membranes in wild-type root tip cells, and was also detected in vesicles. Tomographic modeling of the Golgi revealed that PDI7 was confined to the cis-Golgi, and accumulated primarily at the cis-most cisterna. Shoot apical meristem cells from transgenic plants overexpressing PDI7 exhibited a dramatic increase in anti-PDI7 labeling at the cis-Golgi. When N- or C-terminal fusions between PDI7 and the green fluorescent protein variant, GFP(S65T), were expressed in mesophyll protoplasts, the fusions co-localized with the ER marker, ER-mCherry. However, when GFP(S65T) was positioned internally within PDI7 (PDI7-GFPint), the fusion strongly co-localized with the cis-Golgi marker, mCherry-SYP31, and faintly labeled the ER. In contrast to the Golgi-resident fusion protein (Man49-mCherry), PDI7-GFPint did not redistribute to the ER after brefeldin A treatment. Protease protection experiments indicated that the Trx domain of PDI7 is located within the ER/Golgi lumen. We propose a model where PDI-C isoforms function as cargo receptors for proteins containing exposed cysteine residues, cycling them from the Golgi back to the ER.

Protein Quality Control in the Endoplasmic Reticulum of Plants

“…Some of the PDIs such as PDI5 or PDI6 -members of subfamily PDI-L, which are categorized by the presence of two thioredoxin domains and two redoxinactive thioredoxin domains -represent classical disulfide oxidoreductases as found in mammals and yeast that have chaperone activity in plants (2). Other PDI proteins display a different domain organization with only one thioredoxin domain (PDI-C subfamily) and an additional domain that is found in proteins implicated in Golgi-to-ER retrograde transport (148). The role of these PDI-C proteins in retrieval of native ER-resident proteins -similar to the well-established KDEL/HDEL receptor -or retrieval of escaped non-native proteins that expose free cysteine residues remains to be demonstrated.…”

Development of a GFP biosensor reporter for the unfolded protein response-signaling pathway in plants: incorporation of the bZIP60 intron into the GFP gene

“…Protoplast isolation and transfection was performed using the Tape- Arabidopsis Sandwich protocol 29 as further modified 30 on 4-week-old Arabidopsis plants (WT Col and the PDI9 overexpressor line as previously described 4 ). The enzyme solution (1% cellulase R10, 0.25% macerozyme R10, 0.4 M mannitol, 10 mM CaCl 2 , 20 mM KCl, 0.1% BSA, and 20 mM MES, pH 5.7) was used to digest the tape-treated leaf tissue for 3 hours in light (intensity of 50–60 μmol m −2 s −1 ).…”

“…The W5 solution was then replaced with MMg solution to a density of 2 × 105/mL. 30 The protoplasts were transfected by gently mixing 200 μL of protoplasts (2 × 10 5 /mL) in MMg solution (0.4 M mannitol, 15 mM MgCl 2 , 4 mM MES, pH 5.7) with 20 μL of plasmid DNA solution (containing ~30 μg of each construct, dissolved in water), and 220 μL of PEG solution (40% PEG, 0.2 M mannitol, 100 mM CaCl 2 ). After incubating at room temperature (RT) for 5 min, the transfection step was stopped by adding 1 mL W5 solution.…”

Expression Characterization of AtPDI11 and Functional Analysis of AtPDI11 D Domain in Oxidative Protein Folding

“…PDI-M subgroup has two members, AtPDI9 and AtPDI10 with a 0 -a-b domain arrangement, which is required for pollen viability and normal exine formation in plants subjected to heat stress [21]. Although the expression patterns and enzyme activities of PDI-B (the sole member AtPDI8) and PDI-C subgroup (AtPDI7, AtPDI12, AtPDI13) have been identified, their physiological functions are still unclear [13,22,23]. Recently, using deletion mutation and biochemical analysis, we found that AtERO1 can interact with multiple AtPDIs, and AtPDI-L members mainly serve as an isomerase, while AtPDI-M/S members are more efficient in accepting oxidizing equivalents from AtERO1 and catalyzing disulfide bond formation.…”