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.
Highlights
In 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
The first 22 amino acids of the deduced PDI8 sequence are predicted by SignalP-4.1 to serve as a cleavable signal peptide, with the resulting mature PDI8 protein having a calculated molecular weight of 47.4 kDa and a theoretical pI of 5.01
To gain further insight into the molecular function of PDI8, we examined if PDI8 can functionally complement the E. coli oxidative protein folding mutant, dsbA−
Summary
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. Disulfide bond formation in secretory proteins primarily occurs within the endoplasmic reticulum (ER), and is mediated by members of the protein disulfide isomerase (PDI) family. The classical PDI (represented by PDIA1 in mammals, and PDI1 in Saccharomyces cerevisiae) is a versatile enzyme capable of catalyzing the oxidation, reduction, or isomerization of disulfide bonds in a wide range of substrate proteins in vitro [5], and can assist in protein folding as a molecular chaperone [21, 32]. The classical PDI structure consists of four modular domains in the arrangement a-b-b’-a’, where a and a’ are catalytic domains sharing homology to thioredoxin [9]. In the case of the pancreas-specific human PDI homolog, PDIA2, the b-b’ region is associated with chaperone activity [11]
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