Vigilin Interacts with Signal Peptide Peptidase
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Lu et al. Proteome Science 2012, 10:33 http://www.proteomesci.com/content/10/1/33 RESEARCH Open Access Vigilin interacts with signal peptide peptidase Stephen Hsueh-Jeng Lu1, Amy Hye Won Jeon2, Gerold Schmitt-Ulms2, Seema Qamar1, Roger Dodd1, Beth McDonald1,YiLi1, William Meadows1, Katie Cox1, Christopher Bohm2, Fusheng Chen2, Paul Fraser2 and Peter St George-Hyslop1,2* Abstract Background: Signal peptide peptidase (SPP), a member of the presenilin-like intra-membrane cleaving aspartyl protease family, migrates on Blue Native (BN) gels as 100 kDa, 200 kDa and 450 kDa species. SPP has recently been implicated in other non-proteolytic functions such as retro-translocation of MHC Class I molecules and binding of misfolded proteins in the endoplasmic reticulum (ER). These high molecular weight SPP complexes might contain additional proteins that regulate the proteolytic activity of SPP or support its non-catalytic functions. Results: In this study, an unbiased iTRAQ-labeling mass spectrometry approach was used to identify SPP-interacting proteins. We found that vigilin, a ubiquitous multi-KH domain containing cytoplasmic protein involved in RNA binding and protein translation control, selectively enriched with SPP. Vigilin interacted with SPP and both proteins co-localized in restricted intracellular domains near the ER, biochemically co-fractionated and were part of the same 450 kDa complex on BN gels. However, vigilin does not alter the protease activity of SPP, suggesting that the SPP-vigilin interaction might be involved in the non-proteolytic functions of SPP. Conclusions: We have identified and validated vigilin as a novel interacting partner of SPP that could play an important role in the non-proteolytic functions of SPP. This data adds further weight to the idea that intramembrane-cleaving aspartyl proteases, such as presenilin and SPPs, could have other functions besides the proteolysis of short membrane stubs. Keywords: Signal peptide peptidase, Vigilin, Biochemistry, Intramembrane-cleaving aspartyl protease, Non-proteolytic function, Interactome Background presumably releases the stubs from the ER membrane. Signal peptide peptidase (SPP) [1] is a member of the SPP may also have additional activities in protein control intramembrane cleaving aspartyl protease family that in the ER. For example, SPP is required for the disloca- also includes the presenilins and SPP-like proteases. tion or retro-translocation of MHC Class I molecules in Members of this family share characteristic YD, GxGD the presence of the human cytomegalovirus (hCMV) and PAL motifs [2]. However, the presenilins cleave Type US2 protein [5]. Others have reported that both catalyt- I transmembrane proteins [3], while the SPP-like pro- ically inactive and wild-type SPP may bind and stabilize teases cleave Type II transmembrane proteins [1]. misfolded membrane proteins [6,7]. Amongst the SPP-like proteases, the ER-resident SPP is Preliminary biochemical and biophysical studies by this thought to cleave the membrane-bound stubs of some and other groups [7] have revealed that the SPP-like pro- secreted proteins following proteolysis of the signal pep- teases in general, and SPP in particular, exist in cell tide by the signal peptidase [1,4], and in doing so lysates as higher molecular mass species than would be predicted from the calculated molecular weight of their * Correspondence: [email protected] respective monomeric polypeptides (Figure 1). One po- 1 Cambridge Institute for Medical Research, Department of Clinical tential explanation for this observation is that the SPP Neurosciences, University of Cambridge, Addenbrooke's Hospital, Hills Road, England, Cambridge CB2 0XY, United Kingdom proteins might exist as dimers [8,9] or higher order as- 2Tanz Centre for Research in Neurodegenerative Diseases, and Departments semblies. However, this observation also raises the possi- of Medicine, Laboratory Medicine and Pathobiology, and Medical Biophysics, bility that there may be ancillary proteins which, University of Toronto, 6 Queen's Park Crescent West, Toronto, ON M5S 3H2, Canada although not required for SPP proteolytic activity, may © 2012 Lu et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lu et al. Proteome Science 2012, 10:33 Page 2 of 13 http://www.proteomesci.com/content/10/1/33 as a candidate SPP interacting protein. Reciprocal co- kDa immunoprecipitations confirmed the interaction between HEK293 SPP and vigilin. Additionally, SPP and vigilin co-localized lysate BN-PAGE in restricted cellular domains, co-fractionated biochem- ically and co-migrated as a single band on BN gels. Results 188 Isolation of endogenous SPP complexes 98 SPP dimer 62 SPP and its potential interacting partners were isolated - 49 from native HEK293 cells using a single-step affinity SPP monomer 38 SDS PAGE purification protocol (for a schematic of the protocol 28 see Figure 2A). Specifically, microsomal membrane iso- kDa Probedwith Anti-SPP-CT lates were solubilized in 0.5% N-Dodecyl-β-D-maltoside Figure 1 SPP exists in higher molecular weight complexes. Top (DDM) and subjected to anti-SPP affinity purification panel, HEK293 lysate solubilized in 0.5% DDM and resolved on 16% using anti-SPP-CT antibody, which recognizes the C- Bis-Tris Blue Native-PAGE gels, revealing SPP-containing complexes at terminal amino acids 358–377 of SPP. In order to gen- around 450 kDa, 200 kDa and 100 kDa. Lower panel, lysates resolved erate the negative control with which to identify and on the BN-PAGE were then resolved on the second dimension/SDS- exclude non-specific binders, the capture antibody was PAGE. SDS-stable SPP dimer can be found in all three high molecular weight bands suggesting that the 100 kDa band observed in the pre-blocked with cognate peptide in the control purifi- first dimension is a SDS-stable SPP dimer. cation, and the cognate peptide was further included during the subsequent affinity matrix washing step. The purified SPP proteins were resolved by SDS-PAGE nevertheless modulate its function. We therefore set out and visualized by Western blotting and silver staining to determine whether the high molecular weight SPP (Figure 2B). In eluates obtained following SPP-specific complexes might contain other proteins, and if so, capture, SPP-immunoreactive bands were observed at whether these other proteins modulated the SPP protease 42 kDa for monomeric SPP (predicted to be 47 kDa) activity function of the SPPs. Using an unbiased mass and at 70 kDa, interpreted to be SDS-stable dimers. spectrometry (MS) based approach, vigilin was identified The discrepancy between the actual molecular weight Figure 2 Purification of SPP containing complexes. (A) Schematic showing the protocol for the purification and iTRAQ-based mass spectrometry analysis of the SPP interacting proteins. (B) Purifications were analysed and compared by Western blot with anti-SPP antibody (left panel), demonstrating that the control purification protocol prevented SPP from being purified, and silver staining (right panel), showing the presence of several co-purified proteins that may represent novel SPP interacting proteins. Lu et al. Proteome Science 2012, 10:33 Page 3 of 13 http://www.proteomesci.com/content/10/1/33 and the observed molecular weight on SDS-PAGE is a file 1), while non-specific binders such as the frequently common observation, in particular for membrane pro- encountered protein HSP70, had iTRAQ 117:116 ratios teins, and in part reflects the effect of bound detergent for the signature mass peaks of less than 1.5 (Figure 3A). [10]. However, in the control purification, no SPP signal However, selective co-enrichment (117:116 ratios ≥ 1.5) was observed, demonstrating that the cognate peptide of the ubiquitous 140 kDa RNA-binding protein vigilin was able to block the isolation of SPP. Parenthetically, was observed together with SPP (Figure 3B). Thirteen this method was used because it was much more effective unique peptides were identified from vigilin in total at reducing SPP binding in the control purification than (Figure 3C). siRNA knockdown of SPP (data not shown). The identity of the co-purified proteins and their level of enrichment Vigilin physically interacts with SPP in the cognate-peptide-blocked control equipment were To validate the iTRAQ results, reciprocal co-immuno- then determined by iTRAQ-labelling coupled with mass precipitation experiments from HEK293 cells were spectrometry (Figure 3A). undertaken. As expected, endogenous vigilin was cap- tured when using endogenous SPP as the bait protein iTRAQ-based analysis of the interactome of SPP (Figure 4A). The reciprocal co-immunoprecipitation Inspection of the iTRAQ data revealed a robust enrich- experiments using anti-vigilin antibody was inconclusive ment of SPP (HM13) (for a list of proteins identified and because the SPP bands migrated very closely to the dena- their iTRAQ 117:116 ratios, see Table 1 and Additional tured IgG, making it difficult to differentiate SPP bands A C Vigilin Protein Sequence 1-100 MSSVAVLTQESFAEHRSGLVPQQIKVATLNSEEESDPPTYKDAFPPLPEKAACLESAQEPSGAWGNKIRPIKASVITQVFHVPLEERKYKDMNQFGEGEQ 101-200 AKICLEIMQRTGAHLELSLAKDQGLSIMVSGKLDAVMKARKDIVARLQTQASATVAIPKEHHRFVIGKNGEKLQDLELKTATKIQIPRPDDPSNQIKITG KH domain 1 201-300 TKEGIEKARHEVLLISAEQDKRAVERLEVEKAFHPFIAGPYNRLVGEIMQETGTRINIPPPSVNRTEIVFTGEKEQLAQAVARIKKIYEEKKKKTTTIAV