Arabidopsis protein disulfide isomerase-8 is a type I endoplasmic reticulum transmembrane protein with thiol-disulfide oxidase activity

Abstract

BackgroundIn eukaryotes, classical protein disulfide isomerases (PDIs) facilitate the oxidative folding of nascent secretory proteins in the endoplasmic reticulum by catalyzing the formation, breakage, and rearrangement of disulfide bonds. Terrestrial plants encode six structurally distinct subfamilies of PDIs. The novel PDI-B subfamily is unique to terrestrial plants, and in Arabidopsis is represented by a single member, PDI8. Unlike classical PDIs, which lack transmembrane domains (TMDs), PDI8 is unique in that it has a C-terminal TMD and a single N-terminal thioredoxin domain (instead of two). No PDI8 isoforms have been experimentally characterized to date. Here we describe the characterization of the membrane orientation, expression, sub-cellular localization, and biochemical function of this novel member of the PDI family.ResultsHistochemical staining of plants harboring a PDI8 promoter:β-glucuronidase (GUS) fusion revealed that the PDI8 promoter is highly active in young, expanding leaves, the guard cells of cotyledons, and in the vasculature of several organs, including roots, leaves, cotyledons, and flowers. Immunoelectron microscopy studies using a PDI8-specific antibody on root and shoot apical cells revealed that PDI8 localizes to the endoplasmic reticulum (ER). Transient expression of two PDI8 fusions to green fluorescent protein (spGFP-PDI8 and PDI8-GFP-KKED) in leaf mesophyll protoplasts also resulted in labeling of the ER. Protease-protection immunoblot analysis indicated that PDI8 is a type I membrane protein, with its catalytic domain facing the ER lumen. The lumenal portion of PDI8 was able to functionally complement the loss of the prokaryotic protein foldase, disulfide oxidase (DsbA), as demonstrated by the reconstitution of periplasmic alkaline phosphatase in Escherichia coli.ConclusionThe results indicate that PDI8 is a type I transmembrane protein with its catalytic domain facing the lumen of the ER and functions in the oxidation of cysteines to produce disulfide bonds. It likely plays a role in folding newly-synthesized secretory proteins as they translocate across the ER membrane into the lumen. These foundational results open the door to identifying the substrates of PDI8 to enable a more thorough understanding of its function in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0869-2) contains supplementary material, which is available to authorized users.

Retarded germination of Nicotiana tabacum seeds following insertion of exogenous DNA mimics the seed persistent behavior

“…Among the chaperone proteins, a protein disulfide isomerase spot was also significantly enhanced in VT2eB, compared to the WT ( Table 2 ). This protein showed an active thioredoxin-like domain and an ER resident signal and was involved in introducing disulfide bonds to nascent polypeptides in the ER lumen [ 64 ]. In seeds, the protein disulfide isomerase was found to play a different role related to protein folding (Kim et al, 2012; Kimura et al, 2015), regulation of cysteine protease activity [ 65 ], chaperone activity [ 66 , 67 ], promotion of specific localization of Cys-rich prolaminin in the core of PBs [ 68 ] and regulation of the proportion of various seed proteins, including storage proteins [ 69 ].…”

Targeting of tail‐anchored proteins to Trichomonas vaginalis hydrogenosomes

“…Interestingly, we identified a type I ER protein, TAPDI, with an N‐terminal SP, a C‐terminal anchor domain and a low net positive charge in the flanking regions. Classical PDIs are soluble proteins and PDI8 that has been identified in terrestrial plants is the only known PDI with TMD close to C‐terminus (Yuen et al ., ). We showed that TAPDI protein is inserted into the ER membrane with an N‐terminal domain facing the ER lumen.…”

“…We can speculate that TAPDI evolved from a soluble PDI version and TMD was added later during the evolution to gain new specific function in ER lumen. It has been suggested that plant PDI8 with a C‐terminal TMD may play a role in protein folding as they translocate across ER membrane (Yuen et al ., ). However, we cannot exclude an alternative possibility that some ER type I proteins served originally in the cytosol and at cytosolic side of ER membrane as TA proteins and later gained SP to be targeted to ER lumen.…”

Combined Transcriptome and Proteome Analysis Reveals the Molecular Mechanism by Which ZmPDI Improves Salt Resistance in Rice (Oryza sativa)

“…AtPDI8 in Arabidopsis has been identified as a transmembrane protein located in the endoplasmic reticulum with thioldisulfide oxidase activity. It contributes to the production of disulfide bonds through cysteine oxidation [15]. Arabidopsis AtPDI1 participates in the response to abiotic stresses by engaging in thiol-disulfide exchange reactions.…”