Protein disulphide isomerase family members show distinct substrate specificity: P5 is targeted to BiP client proteins

Abstract

At least 17 members of the protein disulphide isomerase (PDI) family of oxidoreductases are present in the endoplasmic reticulum (ER) of mammalian cells. They are thought to catalyse disulphide formation to aid folding or to regulate protein function; however, little is known about their individual functions. Here, we show that some proteins that enter the ER are clients for single oxidoreductases, whereas others are clients for several PDI-like enzymes. We previously identified potential substrates for ERp57, and here identify substrates for ERp18 and ERp46. In addition, we analysed the specificity of substrates towards PDI, ERp72, ERp57, ERp46, ERp18 and P5. Strikingly, ERp18 shows specificity towards a component of the complement cascade, pentraxin-related protein PTX3, whereas ERp46 has specificity towards peroxiredoxin-4, a thioredoxin peroxidase. By contrast, most PDI family members react with Ero1alpha. Moreover, P5 forms a non-covalent complex with immunoglobulin heavy chain binding protein (BiP) and shows specificity towards BiP client proteins. These findings highlight cooperation between BiP and P5, and demonstrate that individual PDI family members recognise specific substrate proteins.

Fig. 1.

Substrate-trapping mutants of PDI family members form mixed disulphides when expressed in HT1080 cells. Each panel represents results from a particular cell line expressing either wild-type (Wt) or a substrate-trapping mutant (mut) of the PDI family member indicated. Whole-cell lysates were separated by SDS-PAGE under either reducing (R) or non-reducing (NR) conditions. Ectopically expressed protein was visualised by western blot with antibody against the V5 tag. All gels were 7.5% acrylamide except for the ERp18 gel, which was a 7.5-12.5% gradient gel.

Fig. 2.

Resolution of substrates for PDI family members by 2D gel electrophoresis. HT1080 cells expressing either substrate-trapping mutant of ERp18, P5 or ERp46 or wild-type ERp46 were treated with NEM and lysed. Clarified lysates were immunoisolated with an anti-V5 antibody conjugated to agarose beads. Proteins were eluted by boiling in SDS and separated under non-reducing conditions. Gel lanes were excised and reduced with 50 mM DTT and separated in a second dimension. Proteins were visualised by silver staining. Proteins migrating faster in the second dimension than the first dimension were excised from the gel and identified by mass spectrometry. The identities of some of the excised protein spots are as indicated (see Table 1 for details). For P5, a 7.5% gel and for ERp18 and ERp46, a 7.5-12.5% gradient gel was used in both directions.

Fig. 3.

P5 forms a non-covalent interaction with BiP and associates with a BiP client protein. (A) HT1080 cells expressing substrate-trapping mutant of P5 was treated with NEM and lysed. Clarified lysates were immunoisolated with an anti-V5 antibody conjugated to agarose beads. Proteins were eluted by boiling in SDS and separated under reducing (red) or non-reducing (non-red) conditions. The protein bands were excised from the non-reducing gel and the indicated proteins identified by mass spectrometry. (B) HT1080 cells expressing either wild-type (wt) or the substrate-trapping mutant (mut) of P5 were either not treated (-XL) or treated (+XL) with a crosslinking agent. Cells were lysed and V5-tagged P5 immunoisolated using anti-V5 antibody immobilised on agarose beads. The immunoisolate was separated on by SDS-PAGE and western blotted with antibody against BiP. (C) Untransfected HT1080 cells (lane 1) or cells expressing the substrate-trapping mutant of P5 (lanes 2-6) were pretreated with 10 mM DTT (lane 4), 1 mM DPS (lane 5) or untreated (lanes 1, 2, 3, 6). Cells were either lysed in lysis buffer in the absence of NEM (lane 2) or lysed in the presence of NEM (lanes 1, 3-5) or in the presence of NEM and ATP (lane 6). V5-tagged P5 was immunoisolated with V5 agarose. The immunoisolate was separated by SDS-PAGE and western blotted with antibody against BiP. (D) Human immunoglobulin heavy chain was translated in the presence of SP cells prepared from either HT1080 cells (lanes 1 and 8) or cells expressing the substrate-trapping mutants of the PDI family members as indicated (lanes 2-7 and 9-14). Following translation for 2 hours, SP cells were isolated and products of translation were either separated by non-reducing SDS-PAGE immediately (totals; lanes 1-7) or following immunoisolation with V5-agarose (V5-IP; lanes 8-14). Radiolabelled proteins were visualised following autoradiography.

Fig. 4.

Assessing substrate specificity by in vitro translation of Ero1α, β1 integrin or LDLR. mRNA encoding Ero1α (A), β1 integrin (B), or LDLR (C) was translated in the presence of SP cells prepared from either HT1080 cells (lanes 1 and 8) or cells expressing the substrate-trapping mutants of the PDI family members as indicated (lanes 2-7 and 9-14). Following translation for 2 hours, SP cells were isolated and products of translation were either separated by non-reducing SDS-PAGE carried out immediately (totals; lanes 1-7) or following immunoisolation with V5-agarose (V5-IP; lanes 8-14). Radiolabelled proteins were visualised following autoradiography.

Fig. 5.

Mixed disulphides are formed between clusterin, PTX3, Prx-IV and procollagen C-propeptide and specific PDI family members. The translation of clusterin (A), PTX3 (B), Prx-IV (C) and procollagen C-propeptide (D) were carried out as in Fig. 4 with the following modifications: PTX3, Prx-IV and procollagen C-propeptide samples were separated through 7.5-12.5% gradient gels, and Prx-IV samples were separated under reducing conditions.

Fig. 6.

Consequence of blocking the entry of substrates into the calnexin cycle on substrate specificity. The translation of β1 integrin (A), LDLR (B) and clusterin (C) was carried out as in Fig. 4 except that SP cells were pre-treated with castanospermine, which was also present during the translation reaction. Gels were run exactly as described in Figs 4 and 5.

Fig. 7.

Substrate targeted for ERAD forms mixed disulphides with P5. Translation of either wild-type α1-antitrypsin (A) or the NHK mutant of α1-antitrypsin (B) was carried out as described in Fig. 4. The band labelled with an asterisk is the aberrantly interchain disulphide-bonded α1-antitrypsin.