In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1

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In vivo trapping of proteins interacting with the chloroplast CLPC1 chaperone; potential substrates and adaptors Cyrille Montandon, Giulia Friso, Jui-Yun Rei Liao, Junsik Choi, and Klaas J. van Wijk J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00112 • Publication Date (Web): 09 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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Journal of Proteome Research

In vivo trapping of proteins interacting with the chloroplast CLPC1 chaperone; potential substrates and adaptors Cyrille Montandon1, Giulia Friso1, Jui-Yun Rei Liao1, Junsik Choi1, Klaas J. van Wijk1* 1Section

of Plant Biology, School of Integrative Plant Sciences (SIPS), Cornell University, Ithaca,

NY 14853 *corresponding author: Klaas J. van Wijk. [email protected]; Tel: 1-607-2553664 ABSTRACT The chloroplast stromal CLP protease system is essential for growth and development. It consists of a proteolytic CLP core complex that likely dynamically interacts with oligomeric rings of CLPC1, CLPC2 or CLPD AAA+ chaperones. These ATP-dependent chaperones are predicted to bind and unfold CLP protease substrates, frequently aided by adaptors (recognins), and feed them into the proteolytic CLP core for degradation. To identify new substrates and possible also new adaptors for the chloroplast CLP protease system, we generated an in vivo CLPC1 substrate trap with a C-terminal STREPII affinity tag in Arabidopsis thaliana by mutating critical glutamate residues (E374A and E718A) in the two Walker B domains of CLPC1 required for hydrolysis of ATP (CLPC1-TRAP). Based on homology to non-plant CLPB/C chaperones, it is predicted that interacting substrates are unable to be released, i.e. they are trapped. When expressed in wild-type, this CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered. Affinity purification of the CLPC1-TRAP resulted in a dozen proteins highly enriched compared to affinity purified CLPC1 with a C-terminal STREPII affinity tag (CLPC1WT). These enriched proteins likely represent CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants and/or were found as interactions for the adaptor CLPS1, supporting their functional relationship to CLP function. Importantly, affinity purification of this CLPC1-TRAP also showed high enrichment of all CLPP, CLPR and CLPT subunits, indicating stabilization of the CLPC to CLP core interaction and providing direct support for their physical and functional interaction. KEYWORDS: Clp protease, chloroplast, Arabidopsis thaliana, Walker B domains, substrate trapping, mass spectrometry, proteolysis, affinity tagging 1 ACS Paragon Plus Environment

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INTRODUCTION ATP-dependent CLP AAA+ chaperones and their serine protease components are present in bacteria and eukaryotic organelles of bacterial origin, namely mitochondria and plastids, where they degrade a broad range of substrates

1-5.

Substrates are unfolded by these AAA+

chaperones and are then directly delivered into the CLP protease core complexes. There are two main types of CLP chaperones, namely CLPX and CLPA/CLPC, with respectively one and two AAA+ domains 5-6. In eukaryotes, CLPX is located in mitochondria (including humans, plants, algae, etc), whereas CLPA/C in eukaryotes is only found in plastids, including chloroplasts and apicoplasts. Higher plant chloroplasts contain CLPC1, CLPC2 and CLPD and each contain the conserved IGF/L motif (or P-loop) that was shown to be required for docking onto the CLP protease core in bacteria 7-10. The controlling step for CLP protease specificity is provided by the initial recognition of substrates by the AAA+ chaperones, in many cases aided by specific adaptors. These adaptors (also named recognins) deliver specific proteins to the CLP chaperones based on degrons within these substrates, but adaptors can also aid in chaperone priming, activation and oligomerization, reviewed in

11-12.

These adaptors are specific for either CLPX or CLPA/C, and adaptors show

great diversity across species, with some adaptors specific for certain species and/or substrates. Examples of CLPA/C adaptors are MecA and McsB for CLPC in the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus 13-14, the nblA adaptors of CLPC for degradation of phycobilisomes in cyanobacteria 15-17, and the widely conserved CLPS for delivery of N-end rule substrates

18-23.

We recently discovered chloroplast CLPF, a protein unique to plants, which

interacts with chloroplast CLPS1, CLPC, the CLP substrate GLUTAMATE T-RNA REDUCTASE (GLUTR). CLPF appears to play a role as co-adaptor with CLPS1 although its precise function remains to be determined 24. The CLP proteolytic core in Escherichia coli consists of 14 identical CLPP subunits

25.

The chloroplast CLP system is far more diversified and complex, with four different CLPR proteins (R1-4) and five different CLPP proteins (P1, P3-6) all assembled in a single tetradecameric protease core complex

4, 26-27.

Higher plant chloroplasts also contain two

additional CLPT proteins that closely associate with the CLPPR complex, either to activate or to stabilize the complex 28-29. So far, several candidate chloroplast CLP substrates have been identified based on their direct interaction with the CLPS1 adaptor using affinity chromatography with recombinant 2 ACS Paragon Plus Environment

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CLPS1, including four enzymes in the shikimate pathway and GLUTR

23, 30.

Furthermore, the

thylakoid copper transporter PAA2/HMA8, DEOXYXYLULOSE 5-PHOSHATE (DXS) in the chloroplast isoprenoid biosynthesis pathway and PHYTOENE SYNTHASE (PSY) involved in carotenoid biosynthesis were reported substrates of the chloroplast CLP system as well as GENOMES UNCOUPLED 1, (GUN1) in Arabidopsis 31-35. Other candidate substrates have been suggested based on comparative proteomics of a range of clp mutants in Arabidopsis, rice and tobacco, but with this comparative proteomics approach it is hard to distinguish between direct and indirect effects - reviewed in 35-36 and 37-38. An alternative, in vivo approach to identify CLP substrates termed ‘substrate trapping’, has been reported for various bacterial CLP systems, either involving the CLP protease core as the trap or the CLP chaperone 39-42. In the CLP protease core trap approach, the catalytic activity of the CLP core is blocked through site-mutagenesis of the serine residue within the catalytic triad (Ser-His-Asp), resulting in accumulation of substrates within the CLP core central cavity, facilitating their identification by tandem mass spectrometry (MS/MS). The interaction of substrates with AAA+ chaperones CLPB and CLPC is ATP-dependent, whereas the subsequent unfolding and release of the substrate requires hydrolysis of ATP. Therefore CLPB/C mutants that can bind but not hydrolyze ATP will result in trapping of substrates (reviewed in 42). In vivo substrate traps of the unfoldase CLPB (not coupled to CLP proteolysis) and CLPC were generated in respectively E. coli and S. aureus based on mutations in the Walker B domains involved in hydrolysis of ATP 43-46. MS/MS analysis in the affinity eluates of the S. aureus in vivo CLPC trap identified dozens of candidate substrates, including known CLP protease substrates, as well as the CLPC adaptor MecA. In the current study, we successfully generated an in vivo CLPC1 substrate trap with a Cterminal STREPII affinity tag in Arabidopsis thaliana by mutating the conserved and critical glutamate residues in the two Walker B domains of CLPC1 required for hydrolysis of ATP. MS/MS analysis identified a dozen proteins that were highly enriched when compared to a CLPC1-WT control line; these proteins likely represent CLP protease substrates and/or candidate adaptors (including CLPF). A subset of these interactors were also identified using our previous in vitro CLPS1 affinity columns and several interactors over-accumulated in clp mutants. To our initial surprise, the CLPC1 trap also stabilized the interaction with the CLPPR protease core, providing direct support for functional interaction between the CLPC1 chaperone and the CLP protease. 3 ACS Paragon Plus Environment


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EXPERIMENTAL PROCEDURES Plant material and growth The Arabidopsis thaliana (Col-0) T-DNA insertion lines clpc1-1 (SALK_014058) and clpc2-2 (SAIL line SAIL_622_B05 ) are described previously in 23 and see 47-48.

Plants were grown in half-strength Murashige and Skoog (½ MS) plates (1x Gamborg’s B5

vitamin and 0.6% Phytoblend) with or without 2% sucrose or on soil. Plants were grown in a light/dark regime of 10h/14h (short day) or 16/8 (long day), at 22 C, 50% RH and 100 µmol photons.s-1.m-2. Generation of CLPC1-WT and CLPC1-TRAP lines, genotyping and RT-PCR The E374A and E718A mutations were introduced in the coding sequence of CLPC1 (AT5G50920) by overlap extension PCR. In addition, the sequence coding for a C-terminal STREPII-tag was introduced by the reverse primer at the 3’ end. The CLPC1-WT sequence was amplified using the same forward and reverse primers. The inserts were sub-cloned in the gateway entry vector pCR8GW (Invitrogen) and finally inserted by LR reaction in the plant expression binary pEARLYGATE100 (pEG100) vector which carries a 35S promoter and a BAR resistance gene for the selection of the transformants on glufosinate ammonium (BASTA), yielding the vectors pEG100-CLPC1-WT and pEG100-CLPC1-TRAP. The pEG100-CLPC1-WT and pEG100CLPC1-TRAP plasmids were transformed in Agrobacterium tumefaciens G3101 strain by electroporation following the electroporation device’s manufacturer instruction (BioRad). The positive clones were selected on rifampicin (10 µg/ml) and gentamycin (50 µg/ml) (for the agrobacterial strain) and kanamycin (50 µg/ml) (for the plasmid). The presence of the CLPC1WT or CLPC1-TRAP plasmid in positive clones was confirmed by PCR using a forward primer in the 35S promoter and a reverse primer in the CLPC1 insert. A Lysogeny Broth (LB) culture inoculated with a positive pEG100-CLPC1-WT or pEG100-CLPC1-TRAP clone was used to transform either flowering wt or homozygous clpc1-1 (SALK_014058; kanamycin marker) plants by the floral dip method

49.

The plants were selected on ½ MS media and 50 µM glufosinate

ammonium. Genotyping was performed as described previously

50.

To prevent cross reaction,

the reverse primers used for the amplification of the endogenous CLPC1 gene, the CLPC1-WT and CLPC1-TRAP transgene cDNA were designed in the 3’ UTR of the endogenous gene or in the STREPII tag 3’ end of the transgene. For RT-PCR, total RNA was isolated with an RNeasy 4 ACS Paragon Plus Environment

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plant mini kit (Qiagen). The first strand was synthesized from equal amounts of total RNA with Superscript III Reverse Transcriptase (Invitrogen). The number of PCR cycles was 23 for ACTIN2, CLPC1-WT and CLPC1-TRAP or 25 for endogenous CLPC1 and CLPC2; this was optimal to visualize the transcripts. Primer sequences can be found in Table S3. Soluble leaf protein extraction 5-10 g of rosette leaves of 2 months old plants grown on soil under short days were ground in liquid nitrogen. The powder was diluted with 1 ml of extraction buffer (50 mM Hepes-KOH pH 8, 10 mM MgCl2, 75 mM NaCl, 250 µg/ml Pefabloc and 15% glycerol) for each gram of powder and filtered through two layers of Miracloth. The cell debris and the membranes were removed by ultracentrifugation for 1h at 100 000 xg in a SW28 Ti rotor (Beckman). The supernatants containing the soluble leaf proteins were used for SDS-PAGE and immunoblotting or for affinity purification of CLPC1 complexes. SDS-PAGE and Immunoblot analysis For immunoblot analysis, protein aliquots of either total leaf protein or affinity purified complexes were separated on Bio-Rad Criterion Tris-HCl precast gels (10.5-14% acrylamide gradient), followed by transfer to nitrocellulose membrane, stained with Ponceau S, and analyzed by immunoblotting using chemiluminescence for detection. Antisera used were: anti-STREPII (1:5000 dilution; Genescript), anti-CLPC1 and anti-CLPC2 from Professor S. Rodermel

51,

anti-CLPD from Professor K. Nakabayashi, anti-GLUTR

(Agrisera, Sweden, #AS10689), and several sera produced by the van Wijk lab against purified recombinant proteins, namely anti-CLPR2

52

, anti-CLPP6

28,

anti-CLPF

23,

anti-CLPS1

23.

Protein concentrations were determined using the BCA Protein Assay Kit (ThermoFisher). Affinity purification of CLPC1-WT and CLPC1-TRAP The soluble leaf proteomes were passed through 0.5 ml beads of StrepTactin or StrepTactin XP (IBA Life Sciences). The resin was washed 2 times with 5 column volumes and the bound recombinant proteins were eluted with 6 column volumes of 2.5 mM desthiobiotin or biotin. The eluates were concentrated about 100 times for further analysis using centrifugal filters (Amicon Ultra 4, 3 kDa cutoff, MilliporeSigma). Light blue Native PAGE of affinity purified complexes For native PAGE analysis of the CLPC1-WT and CLPC1-TRAP eluates, protein aliquots of the CLPC1-WT and CLPC1-TRAP 5 ACS Paragon Plus Environment


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affinity eluates were separated using the NativePAGE Novex gel system (Invitrogen) with precast 4-16% acrylamide Bis-Tris gels (Invitrogen)

27.

This was followed either by immunoblot

analysis as described above, or MS/MS analysis as described below. Proteomics and Mass spectrometry Affinity eluates of CLPC1-WT and CLPC1-TRAP were separated by SDS-PAGE on Biorad Criterion Tris-HCl precast gels (10.5-14% acrylamide gradient) with two biological replicates (#1 and #2). Biological replicates consisted of completely different batches of plants (independently grown) followed by standard affinity purification protocols and subsequent sample processing. Aliquots of the CLPC1-WT and CLPC1-TRAP affinity eluates of replicate #2 were also separated by light blue native gel electrophoresis. Each of the SDS-PAGE and native PAGE gel lanes were completely cut into consecutive gel slices, followed by reduction, alkylation, and in-gel digestion with trypsin as described in great detail in 53,

essentially as follows. The gel bands were cut into 1x1 mm pieces and washed 3 times in

water (dd), ii) dehydrated in 100% acetonitrile, and then iii) incubated for 30 min at 56 °C in 10 mM dithiothreitol (DTT) and 0.1 M NH4HCO3, followed by iv) alkylation in 55 mM iodoacetamide, 0.05 M NH4HCO3. The gel pieces were then collected by centrifugation, washed in 0.1 M NH4HCO3 and 50% acetonitrile, and finally digested overnight at 37°C in 12.5 ng/μl trypsin, 10 mM NH4HCO3 and acetonitrile. The resuspended peptide extracts were analyzed using a LTQOrbitrap (the first LTQ-Orbitrap generation – 2005) interfaced with a nanoLC system (two Surveyor MS pumps) and MicroAS Autosampler as purchased in 2005 from ThermoFischer Scientific. Peptide samples were automatically loaded on a guard column (C18 PepMap 100, 5μm, 100A; 300 μm i.d. x 1 mm- Thermo Scientific) via an autosampler followed by separation on a PepMap C18 reverse-phase nanocolumn (Inertsil ODS-3, 3 μm C18; 75 μm i.d. x 15 cm; Thermo Scientific) using 85 min (native page samples) or 120 min (SDS-PAGE) gradients with 90% water, 5% DMSO, 5% ACN, 0.1% FA (solvent A) and 90% ACN, 5% DMSO, 5% water, 0.1% FA (solvent B) at a flow rate of 200 nl/min. Two blanks were run after every sample. The acquisition cycle consisted of a survey MS scan in the Orbitrap with a set mass range from 350 to 1800 m/z at the highest resolving power (100,000) followed by five data-dependent MS/MS scans acquired in the LTQ. Dynamic exclusion was used with the following parameters: exclusion size, 500; repeat count, 2; repeat duration, 30 s; exclusion time, 180 s; exclusion window, ±6 ppm. Target values were set at 5 x 105 and 104 for the survey and tandem MS scans, respectively. Mass window for precursor ion selection was set at 2 m/z with monoisotopic peak 6 ACS Paragon Plus Environment

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selection and the FTMS preview option was used. MS survey scans in the Orbitrap were acquired in one microscan. Fragment ions spectra were acquired in the LTQ as an average of two microscans. Peak lists (in .mgf format) were generated from RAW files using DTA supercharge software, and all .mgf files were recalibrated as in

53.

Recalibrated files were searched with

MASCOT v2.2 against TAIR10 released in 2011 (https://www.arabidopsis.org/download/indexauto.jsp?dir=%2Fdownload_files%2FMaps%2Fgbrowse_data%2FTAIR10) including a small set of typical contaminants and the decoy (71,148 sequences; 29,099,536 residues). Three parallel searches (Mascot p-value .05) (Fig. 3B). Comparing the segregating wt/CLPC1-TRAP progeny on agar plates with glufosinate (BASTA) showed virescent and albino 10 ACS Paragon Plus Environment

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plants, in addition to tiny seedlings that could not grow. However on plates without glufosinate, plants segregated in virescent, albino and wt plants (Fig. S2). Moreover, albino plants showed a higher STREPII protein level than in the virescent plants (Fig. 2B) consistent with the presence of two CLPC1-TRAP alleles. The virescent and albino phenotypes in the wt/CLPC1-TRAP lines are therefore likely due to a dominant negative effect of the TRAP mutations. It follows that the stronger phenotype of the homozygous albino wt/CLPC1-TRAP plants is a dosage effect of the defective CLPC1-TRAP protein.

Accumulation of the CLP machinery in the CLPC1-TRAP, CLPC1-WT Before using the homozygous wt/CLPC1-WT and heterozygous virescent wt/CLPC1-TRAP lines for CLPC1 interaction studies (Fig. 4A), we determined the accumulation levels of the CLP chaperones, representative subunits of the CLP protease cores (CLPR2 and CLPP6), the adaptor and coadaptor ClpS1 and CLPF, and the CLP substrate GLUTR. Figure 4A shows examples of plants of both genotypes used for this analysis. Total soluble leaf proteomes were isolated and run out on SDS-PAGE gels, followed by blotting and immuno-detection. Fig 4B shows an example of a Ponceau stained blot of the soluble proteomes of the two genotypes and the two proteomes appear very similar based on the staining pattern. Immunoblotting with specific antisera is shown in Fig. 4C. This showed that accumulation levels of tagged CLPC1 (using the STREP serum) as well as the sum of endogenous CLPC1 and transgenic CLPC1 (using the CLPC1 serum) were 11 ACS Paragon Plus Environment


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comparable across the two lines. However, CLPC2 was clearly several fold higher in the wt/CLPC1-TRAP line than in the wt/CLPC1-WT line. This is consistent with the strong upregulation of CLPC2 in the clpc1-1 null mutant

23

and indicates that the expression of the

CLPC1-TRAP protein is likely perceived as CLPC1 chaperone limitation.

Representative subunits of the CLP protease core (CLPR2 and CLPP6) and CLPF were ~2 fold higher in wt/CLPC1-TRAP, whereas CLPS1 was >2 fold higher in the wt/CLPC1-TRAP line. CLPS1 also showed a strong upregulation in the clpc1-1 line

23,

again reinforcing the notion of

limited CLPC1 chaperone capacity in the CLPC1-TRAP line. In contrast, accumulation levels of the CLPD chaperone and CLP substrate GLUTR were the same in the two transgenic lines. The CLPD is a senescence and drought-induced chaperone 37, 56-59 and likely plays a more specific role in protein homeostasis than CLPC1 and CLPC2. We note that the CLPD antiserum showed an increased response from the 1 μg to the 10 ug lane, but it did not further increase in the 20 μg lane. This is due to the limited titer and amount of serum available. The CLP substrate GLUTR 12 ACS Paragon Plus Environment

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did not overaccumulate in the wt/CLPC1-TRAP line; this is not surprising since we measure here steady state levels, and GLUTR is under multiple levels of regulation. Degradation is particularly pronounced during extended dark treatment

30.

Furthermore, the TRAP line still has CLP

chaperone and protease capacity and this still will degrade substrates, albeit likely with slower dynamics. Successful affinity purification of CLPC1-WT and CLPC1-TRAP We then used the wt/CLPC1-TRAP heterozygous line for affinity purification of CLPC1 to discover potential interacting proteins representing candidate substrate and novel adaptors. The wt/CLPC1-WT line was used as a control for unspecific interactors, assuming that substrates do not significantly accumulate (or at much lower levels) with fully functional CLPC1 chaperones. The STREPIItagged CLPC1 proteins were purified on streptactin columns following protocols used for determination of the CLP core subunit composition 27-28. Aliquots of protein eluates were run out on SDS-PAGE gels on the basis of elution volume, and then blotted, stained with Ponceau (Fig. 4D), and then probed with antisera for the same set of CLP proteins and GLUTR (Fig. 4E) used to the total soluble leaf extracts, as discussed above. An example of a Ponceau stained blot showed a clear band just below the 100 kDa marker, corresponding to the STREPII–tagged CLPC1-TRAP and CLPC1-WT proteins, but other protein bands are not (easily) visible (Fig. 4D). Immunoblots showed similar levels of STREPII-tagged CLPC1 and total endogenous and tagged CLPC1, perhaps both a bit higher in the CLPC1-WT aliquots (Fig. 4E). CLPC2 and CLPF, and in particular CLPR2, CLPP6, CLPS1 were all several fold enriched in the CLPC1-TRAP eluates. In contrast, levels of CLPD and GLUTR were slightly lower in the CLPC1-TRAP (Fig. 4E). We then carried out two additional and independent affinity purifications, followed by SDS-PAGE, in-gel tryptic digestion and extraction of peptides, which were then identified and quantified by MS/MS analysis. Identified proteins were annotated based on curated information in the Plant Proteome DataBase (PPDB). The identified proteins and associated information are summarized in Table S1, and available via PPDB and the PRIDE public data repository 60. This identified some 500 proteins, of which nearly 50% were annotated plastid proteins. Crosscorrelation between relative protein abundances in eluates of CLPC1-WT and CLPC1-TRAP is shown in Fig. 5A. Based on peptides matching to CLPC1 and/or CLPC2, the CLPC1,2 protein family was by far the most abundant protein in the eluates indicative of successful affinity 13 ACS Paragon Plus Environment


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purification; on average 28% of all adjusted spectral counts (adjSPC – the adjustment was to take into account shared peptides between proteins – see

53)

were from endogenous and

transgenic CLPC1,2. Other abundant proteins were the endogenous enzymes that have biotin as a co-factor (ACC1,2 and MCCA and MCCB) and therefore bind to the streptavidin affinity matrix, as well as highly abundant stromal proteins RBCL and its activase (RCA), elongation factor Tu, and THI1 involved in thiamin biosynthesis (Fig. 5A). ‘Error-tolerant’ MS/MS searches using MASCOT (http://www.matrixscience.com/) allow for amino acid substitutions 54, 61. Using such error-tolerant searches, we specifically confirmed the two Glu to Ala mutations (E374A and E718A) in CLPC1-TRAP protein with a high number of matched

MS/MS

spectra

in

QSDEIILFIDE374VHTLIGAGAAEGAIDAANILKPALAR,

the and

tryptic

peptides

RPYTVVLFDE718IEK

and

RRPYTVVLFDE718IEK (Fig. S3). Since the CLPC1-TRAP was expressed in the wt background, we also found the equivalent peptides without these mutations matching against CLPC1 indicating that the CLPC1-TRAP protein formed mixed oligomers with endogenous CLPC1 (Fig. S3). This is expected since there are no reasons to assume that the Walker B mutations affect CLPC structure or oligomerization 42 (also based on information for the Walker B mutant in CLPB 14 ACS Paragon Plus Environment

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homologs 44, 46 whereas the successful complementation of clpc1-1 with the CLPC1-WT shows that the C-terminal STREPII tag does not affect CLPC assembly. The immunoblots showed that CLPC2 but not CLPD was enriched in the CLPC1-TRAP. To evaluate if this pattern was also found in the MS/MS data, we analyzed the identified peptides matched to CLPC1, CLPC2 and CLPD. Because CLPC1 and CLPC2 are very closely related (Figure S4), many peptides are shared but we did find unique peptides for CLPC2 in both CLPC1-TRAP and CLPC1-WT supporting mixed CLPC1,2 oligomers or trapped CLPC2 proteins. CLPD and the CLPC isoforms have about 45% sequence identity; we identified 21 peptides matched to CLPD of which 12 were unique to CLPD (Figure S5), most of them in one of the CLPC1-TRAP replicates. This compares to >9000 peptides matching to CLPC1/CLPC2 across the eluate samples, indicative of a highly sub-stoichiometric accumulation of CLPD in the CLPC1 eluates (Table S1) (see DISCUSSION). The CLP core complex co-purifies with the CLPC1-TRAP; stabilized CLP core to CLPC1 interactions All eleven CLPPRT core proteins (CLPP1,3-6, CLPR1-4 and CLPT1,2) were identified in both CLPC1-TRAP eluates (Table 1; Table 1A). In contrast, the CLP core proteins were either not identified in the CLPC1-WT eluates or with much less MS/MS spectra (Table 1; Fig. 5A). The CLP core complex including CLPT1,2 was on average 11-fold enriched when normalized based on total amount protein (not shown) and 6-fold when normalized to CLPC (Fig. 5B). The ratio between the relative proportions of the P-ring, the R-ring and CLPT1,2 was similar across the CLPC1-TRAP and CLPC1-WT eluates (Fig. 5B). Interestingly, the co-adaptor CLPF 24 was identified with high sequence coverage and multiple different tryptic peptides in the eluates of CLPC1-TRAP (with 34 MS/MS spectra) but with just 6 MS/MS spectra for CLPC1-WT (Table 1; Fig. S6). The adaptor CLPS1 23 was not identified in any of the affinity eluates, but we note that due to its small size (12 kDa) and low number of predicted tryptic peptides, CLPS1 is relatively difficult to identify by MS/MS. Indeed, in the independent affinity purification for the immunoblot analysis in figure 4D,E, we observed a relatively strong enrichment of both CLPS1 and CLPF. Native gel electrophoresis followed by MS/MS of the CLPC1-TRAP eluates, but not CLPC1-WT eluates, showed that subunits of the CLP core migrated in two peaks, one around 350 kDa indicative of the intact CLP core, and also at a lower molecular mass range (Fig. 6A). For comparison, the 550 kDa RUBISCO holocomplex migrates in higher molecular weight 15 ACS Paragon Plus Environment


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fractions than the CLP core subunits (Fig. 6A). The CLPC1-WT and CLPC1-TRAP were detected at around 200 kDa indicative of dimers (Fig. 6A) and consistent with our previous observations of stromal endogenous CLPC1,2 on native gels 62. Immunoblots with anti-STREPII were consistent with the MS/MS results showing the STREPII tagged CLPC1-WT and CLPC1TRAP migrating as ~200 kDa complexes (Fig. 6B). Some weaker signals in higher mass regions indicate the presence of CLPC1-STREP in larger complexes.

Candidate CLPC1 substrates Based on the conservation of the Walker B domains between CLPC1, SaCLPC and EcCLPB and prior substrate trapping results for these two homologs 43-46, the E374A and E718A mutations in the CLPC1-TRAP protein will block ATP hydrolysis and likely generate stable interaction of CLPC1-TRAP with its substrates. We therefore evaluated enrichment of other proteins in CLPC1-TRAP as compared to CLPC1-WT; proteins identified based on only one peptide (even if observed multiple times) were discarded to reduce possible false positive identifications and to reduce the number of possible unspecific interactors. There were 21 proteins detected in both CLPC-TRAP replicates but not detected in CLPC-WT (Table S2); four of these were also observed in the native PAGE for CLPC1-TRAP, but not CLPC1-WT. Of these 21 proteins, 14 were confirmed plastid proteins (based on curated assignment in PPDB) 16 ACS Paragon Plus Environment

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and 3 are likely plastid proteins because they have predicted chloroplast transit peptides or other attributes. To evaluate the likelihood that these 17 (likely) chloroplast proteins were CLPC1 substrates, we: i) calculated their relative abundance in the samples, either based on overall abundance or normalized to CLPC1, ii) mapped the observed tryptic and semi-tryptic peptides and sequence coverage for each protein and possible orthologs and evaluated possible PTMs, iii) verified if proteins were found to be upregulated in our prior comparative proteome analysis of clp mutants clpc1-1 interactor

23,

23,

clps1

23,

clpc1-1 clps1

23,

clpr2-1

63

or clpp3-1

64

or identified as a CLPS1

iv) searched for functional information for each of these proteins. This information

is summarized in Table 2, whereas protein sequence coverage information (based on peptides from matched MS/MS spectra) is provided in the Figure S7 and other details provided in Table S2. We discuss these proteins in the following paragraphs. The protein AT1G48450 was identified with the most MS/MS spectra; it has no known function and contains a DUF760 domain (Table 2). Mapping of the identified peptides of this protein in the CLPC1-TRAP showed very high sequence coverage for nearly the complete intact mature protein (Fig. S7). Previously we identified this protein in comparative proteomics experiments of total leaf extracts of clpr2-1 but not in wt 63. AT1G48450 has four homologs with predicted cTP or mTP, and one of them (AT3G17800) was also found in both CLPC1-TRAPs with a total of 39 matched MS/MS, but not CLPC1-WT (Table 2). DUF179-containing protein AT3G29240 has no known function and was also identified with a high number of spectra in both replicates (Table 2). We identified its likely N-terminus starting after a cysteine residue (C↓SLS) (Fig S7); we note that a mature N-terminus starting immediately after a cysteine residue is quite common for chloroplast stromal proteins protein was previously identified in clpr2-1 but not the parallel wt samples enriched in clpc1, as well as clpc1 clps1

23.

63,

65.

This

and was highly

AT3G29240 has two homologs that also have a

predicted cTP and one of them, AT1G33780, was found in equal amounts in CLPC1-WT and CLPC1 TRAP (Table S1), whereas we never identified the homolog AT3G19780 (see PPDB). Chloroplast 5-amino-6-ribitylamino- 2,4(1H,3H) pyrimidinedione 5′-phosphate (ARPP) phosphatase 2 (FHy2 or PYRP2; AT4G11570) identified in both CLPC1-TRAP replicates is involved in riboflavin biosynthesis 66. PYRP2 over-accumulated in clpc1 clps1, clpc1, as well as clps1 and it was also observed as a CLPS1 interactor

23.

PYRP2 is member of the large halo-

17 ACS Paragon Plus Environment


Page 18 of 40

acid dehydrogenase (HAD) superfamily. PYRP2 has one close homolog AT3G10970 (without ARPP phosphatase activity 66 which was also enriched in the CLPC1-TRAPs (Table 2). Three plastid 5'-adenylylsulfate reductases (APR1,2,3) involved in sulfur assimilation

67

were identified in the CLPC1-TRAP. APR2 was several fold more abundant compared to the other chloroplast APR homologs, and we identified both the N- and C-termini of APR2 and no obvious PTMs (Fig. S7). EXECUTER2 (EXE2) is a UVR domain containing protein involved in single O2 damage and retrograde signaling

68;

however its function is not understood. Importantly EXE2 was

previously found enriched in leaf extracts of all three replicates of clpc1-1 and clpc1-1 clps1, but not in wt 23, as well as in two replicates of clpr2-1 63. The close homolog EXE1 was not identified in any of the eluates, neither in CLPC1-WT nor CLPC1-TRAP. EXE2 seems a good candidate for a specific adaptor, given its role in signaling and the presence of the UVR domain that is also present in both CLPC1 and CLPC2 (but not CLPD), as well as CLPF

24.

Alternatively, it could

also be a CLP substrate, similar as EXE1 was suggested to be a substrate for the thylakoid FTSH2 protease 69. We also identified three relatively abundant metabolic enzymes (E2 of the pyruvate dehydrogenase (PDH) complex, isopropylmalate isomerase (IPMI) and glucose-6-phosphate 1dehydrogenase 1 (G6PD-1), each with relatively low numbers of MS/MS spectra. Finally, AT5G52960 (no known function) was identified in both CLPC1-TRAP eluates; previously this protein was identified as a specific interactor to CLPS1

23.

Interestingly, this protein was found

in multiple phosphoproteomics studies based on the phosphorylation within the tryptic peptide SSDAEEVSDTEDEWLK (see PhosPhAtDB). Additionally, 12 proteins were identified in one of the two traps but not in CLPC1-WT; ten of these proteins are plastid-localized (Dataset 1B). The two proteins identified with the highest number of MS/MS spectra (10 and 12) and each with multiple different peptides, are a plastid protein with unknown function (AT5G24060), and stromal shikimate dehydrogenase (DHS) (AT4G33510) that we previously identified as a specific interactor of CLPS1 23 (Table 2). Since CLPC1-WT also interacts with substrates, even if transiently, we also calculated relative enrichment of all proteins present in both CLPC1-TRAP samples as well as in one or both CLPC1-WT. The highly abundant THI1, involved in chloroplast thiamine synthesis, stands out as a protein 5-6 fold enriched in the CLPC1-TRAP eluates compared to CLPC1-WT, both when normalized to the total adjSPC, and when normalized based on CLPC1 (Fig. 5A; Table S1). We 18 ACS Paragon Plus Environment

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note that THI has a very high turnover rate

70

and was previously suggested to be a CLP

substrate 4, 35. DISCUSSION The primary objective of this study was to identify additional candidate substrates and possibly adaptors of the chloroplast CLP protease system by generating an in vivo CLPC1 trap in Arabidopsis. We used mutations in conserved glutamate residues in both Walker B domains (E374A and E718A) since previous studies for bacterial CLPB and CLPC firmly established that mutagenesis of these conserved residues results in stable associations between the CLP chaperones and their substrates due to loss of the capacity to hydrolyze ATP, while not affecting the actual binding of ATP

43-44, 46, 71.

When expressed in Arabidopsis wild-type, this chloroplast

CLPC1-TRAP induced a dominant visible phenotype, whereas no viable mutants that express CLPC1-TRAP in the clpc1-1 null mutant could be recovered, most likely because this resulted in embryo lethality. Affinity purification of this CLPC1-TRAP, but not CLPC1-WT, showed high enrichment of the CLP core complex indicating stabilization of the CLPC to CLP core interaction (see below in the section ‘In vivo CLPC1 interaction to the chloroplast CLPPR core’). This provides direct support for functional interaction which is important since co-purification of CLP core and its chaperones has proven difficult in the past. We discuss reasons why the expression of the CLPC1-TRAP induces such a strong phenotype in the subsequent section ‘The dominant phenotype of the CLPC1-TRAP transgene’. Finally, affinity purification of the CLPC1-TRAP resulted in a dozen highly enriched proteins, likely representing CLP protease substrates and/or new adaptors. Several of these trapped proteins over-accumulated in clp mutants, supporting their functional relationship to CLP function, as discussed below (section ‘Identification of putative CLP substrates and adaptors’). We note that there is a potential caveat for the substrate identification in that the CLPC1-TRAP line is smaller, virescent and delayed in growth compared to the CLPC1-WT control line (although we note that this phenotype is less pronounced than the homozygous clpc1-1 phenotype

23).

Proteins enriched in the CLPC1-TRAP eluates could be

non-specific contaminants and enriched compared to CLPC1-WT because they overaccumulate in chloroplasts as part of the phenotype (directly or indirectly due to reduced CLP capacity) rather than because they were trapped as substrates in the CLPC1-TRAP chaperone complex. However, given the very high sequence coverage and the repeat observations, this alternate explanation seems less likely. 19 ACS Paragon Plus Environment


Page 20 of 40

A model for multi-step CLP function Clp dependent proteolysis is an ATP-dependent multistep, regulated process that involves not only CLP chaperones and CLP proteases, but often also one or more adaptors (and even anti-adaptors)

5-6, 12, 72-73.

ATP binding and hydrolysis is

required for substrate unfolding, and in case of CLPC/A homologs also for chaperone oligomerization. In contrast the actual proteolytic cleavage by the catalytic CLP protease core does not require ATP, but specific and dynamic interactions (docking) between the CLPC/A hexamer and the CLP protease core is required for catalytic cleavage

2-3.

Figure 7 provides a

schematic model of this multi-step process mostly based on studies of bacterial CLPA/P systems, and serves to help interpret and discuss the experimental findings in the current chloroplast CLPC1 trapping study.

The resting state of CLPX chaperones is typically a hexamer, whereas the resting state of CLPA/C chaperones is a dimer requiring regulated hexamerization prior to binding to the barrel-shaped tetradecameric protease CLP protease core complex – reviewed in 7 (Fig. 7). The CLPA/C hexamerization requires ATP and frequently also adaptors, as recently demonstrated 20 ACS Paragon Plus Environment

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in structural detail for the gram-positive bacterium S. aureus MecA and CLPC

14.

The resting

state of chloroplast CLPC1,2 is clearly a dimer as demonstrated by native electrophoresis and gel filtration of stromal proteomes of Arabidopsis

24, 62.

It is not known if chloroplast CLPC

hexamerization does require interactions with adaptors and/or substrates. Purified recombinant CLPC2 and CLPD were reported to also formed higher order assemblies (~500-700 kDa) in the presence of ATP 74; in that scenario there were no adaptors nor specific substrates present but it is hard to judge the physiological significance of such recombinant oligomerization results. Following formation of the CLPC hexameric rings, they can dock onto the tetradecameric CLP core complex. In case of E. coli and many other bacteria, the tetradecamer is homomeric since there is only one CLPP gene, and it is believed that CLPA or CLPC can bind to either adaxial side. However in case of chloroplasts, the CLP core is asymmetric with a P-ring and an R-ring, with docking of CLPC hexamers on the R-ring (consisting of CLPP1 and CLPR1-4) and not the P-ring 4. The two plant-specific CLPT proteins are believed to primarily interact with the P-ring 28.

Following the formation of the CLPC-CLP protease oligomer, proteolysis can start and

requires ATP hydrolysis for protein unfolding by the CLP chaperone. Presumably, each CLPCCLP protease assembly can engage in the (sequential) degradation of multiple substrates. When no new substrates are engaged with the complex, the CLPC-CLP protease assembly dissociates, completing the cycle. In vivo CLPC1 interaction to the chloroplast CLPPR core The MS/MS analysis identified all nine CLPPR subunits, as well as CLPT1,2 in the CLPC1-TRAP eluates. Moreover, native gel electrophoresis showed that most of the CLPP and CLPR subunits were part of the typical 350 kDa core complex, whereas CLPC resolved as ~200 kDa dimers. Furthermore, immunoblotting of the affinity eluates specific CLP antisera confirmed the strong enrichment of the CLP core in the CLPC1-TRAP eluates. Based on the general knowledge of CLPA/C-P protease systems 7, it is most likely that the inability to hydrolyze ATP prevented the unfolding of substrates but not CLPC hexamerization and docking onto the CLP core, with the block in substrate unfolding stabilizing the CLPC1-CLP core-substrate complex. The binding of the substrate might promote the interaction of the CLPC1 hexamer with the core, or alternatively, the docking of the CLPC1 hexamer to the CLP core promotes the recruitment of the substrates. The CLPC-CLP core interaction clearly was not sufficiently stable to maintain its interaction during the elution from the affinity column and/or native gel separation. Studies to stabilize the CLPC-CLP core complex 21 ACS Paragon Plus Environment


Page 22 of 40

using crosslinking and size separation by alternative techniques might better preserve this large assembly (estimated at >950 kDa). There have been previous studies aimed at identifying chloroplast CLPC-CLP protease core interactions. Previous studies of chloroplast stroma using import of radiolabeled CLP precursors, co-immunopreciptation and/or column chromatography did suggest an ATPdependent association between CLPC and CLPP 75-77. However, this was prior to the recognition that the chloroplast CLP protease complex consists of a mixture of different P, R and T subunits. We spent considerable time and efforts in the past to reconstitute wild-type

Arabidopsis

chloroplast CLPC1-CLP core interactions from stromal extracts using e.g. non-hydrolyzable ATP analogs and gel filtration chromatography, but we were not able to observe significant interactions. Furthermore, affinity purifications using stable Arabidopsis transgenic STREPIItagged CLPT1, CLPT2, CLPR4 and CLPP5 followed by mass spectrometry did not identify significant amounts of CLPC or CLPD

27-28

in the eluates, further supporting the notion that the

CLPC-CLPPR core interaction is transient and/or unstable. The current strong and consistent co-purification of all CLPPRT subunits with the CLPC1-TRAP is therefore an important confirmation of the functional partnership between CLPC1 and the CLPPR protease core complex in chloroplasts. The dominant phenotype of the CLPC1-TRAP transgene The CLPC1 null allele shows a pale green phenotype throughout development, whereas CLPC2 and CLPD null alleles have no visible phenotypes

23, 51, 55.

Complete loss of both CLPC1 and CLPC2 proteins but also loss of

the CLPP5 protease core subunits results in embryo lethality

48, 78

whereas other CLPPR core

subunits results in seedling lethality 78. These loss-of function alleles demonstrate that both the total CLPC1,2 chaperone capacity and the CLPPR protease core capacity are required for plant growth and development; loss of either capacity is detrimental for the plant. In all cases, we must assume that loss of CLP capacity results in accumulation of dysfunctional proteins, protein fragments and/or protein aggregates with high toxicity for the plastid. The heterozygous wt/CLPC1-TRAP allele is smaller and paler than wt, but less so than the clpc1-1 null allele. However the homozygous CLPC1-TRAP plants have a far more severe phenotype than clpc11 indicating a loss of function beyond CLPC1 itself.

Based on this knowledge, and the

observation of endogenous CLPC1 and CLPC2 in the CLPC1-TRAP eluates (by MS/MS and immunoblotting with CLPC1 and CLPC2 specific antisera), we postulate two main explanations 22 ACS Paragon Plus Environment

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for the phenotypes of the heterozygous (virescent) and homozygous (albino and dwarf) wt/CLPC1-TRAP lines: i) the CLPC1-TRAP forms mixed oligomers with endogenous CLPC1 (since the TRAP line is in a wt background) and endogenous CLPC2, thereby reducing total CLPC unfolding capacity and hampering substrate degradation by the CLP protease core. This assumes that hexamers containing a mixture of inactive CLPC1 and endogenous (functional) CLPC1,2 proteins are not able to unfold CLP substrates effectively. ii) the CLPC1-TRAP recruits and stably associates with CLP protease cores into a non-functional complexes thereby reducing CLP protease capacity below the minimal threshold for survival. Residual endogenous CLPC1 and/or CLPC2 are essentially outcompeted by transgenic CLPC1TRAP for association to the CLPPR core. These explanations are also consistent with our inability to recover a clpc1-1/CLPC1-TRAP line. In this situation, there is no endogenous CLPC1 but only CLPC2, which is known to be expressed at far lower levels than CLPC1 even if CLPC2 levels are upregulated (see 4). Consequently, CLPC1-TRAP could simply outcompete CLPC2 for association to the CLPPR core, which is further aggravated if CLPC1-TRAP forms inactive mixed CLPC hexamers with CLPC2. Identification of putative CLP substrates and adaptors A dozen or so CLP protease substrates have been identified or inferred from a range of experimental papers - reviewed in 4 and recent studies 30, 32, 38. So far, two adaptors have been identified in chloroplasts, namely the well-conserved CLPS1 protein and CLPF, a protein unique to higher plants. MS/MS analysis of affinity purified CLPC1-TRAP and CLPC1-WT identified some 17 proteins only found in the CLPC1-TRAP (Table 2). The MS/MS analysis did not suggest any particular PTM and for none of these proteins there was good evidence that they were already cleaved by other proteases and on their way to be degraded. The exception is CPN10 since we only detected peptides matching to the C-terminal region, suggesting that a C-terminal fragment of CPN10 was trapped (Fig. S7). Two of the 17 proteins (unknown AT5G52960 and DAP synthase 2) were identified among the nine specific interactors to CLPS1 23. Six proteins (DUF 179 - AT3G29240, unknown AT1G63610, FHy2/PYRP2, EXE2, E2-subunit of PDH, DAHP synthase 2) overaccumulated in clpc1, clpc1 clps1 or clps1 null mutants 23 which could reflect i) reduced degradation rate due to



Page 24 of 40

loss of CLP degradation, ii) increased expression as a response to protein (folding) stress, or iii) indirect (pleiotropic) effects on protein accumulation. Those proteins that have enzymatic functions in metabolic pathways (FHy2/PYRP2, HAD, APR1,2,3, E2-PDH, IPMI, G6PD-1 and DHS2) or other well documented functions (CNP10 and 30S ribosomal proteins S7A,B) are unlikely to be adaptors or other types of direct regulators of CLP activity; instead their enrichment suggests that they are possible CLPC1 substrates. These enzymes each belong to different major metabolic pathways (Table 2), namely riboflavin (vitamin B2) biosynthesis (FHy2/PYRP2), sulfate metabolism (APR1,2,3), fatty acid metabolism (E2PDH), leucine synthesis (IPMI), the oxidative pentose phosphate (OPP) pathway (G6PD1) and the shikimate pathway (DHS2). Given that DHS2 was also a strong and specific interactor to the adaptor CLPS1, along with other enzymes in the shikimate pathway, DHS2 is a particular strong CLP protease candidate – see for references and discussion in

23.

Prior studies have

shown that the chloroplast CLP system is important in regulation of chloroplast metabolism, with a number of metabolic proteins shown to be candidate or confirmed CLP substrates

35.

Our

current trapping study further expands the list of candidate chloroplast CLP substrates. EXE2, but not its homolog EXE1, was also observed in all stromal proteome replicates of clpc1-1 and clpc1-1 clps1

23

and also in two replicates of clpr2-1

63

but not in wt. Furthermore,

the peptides identified for EXE2 in the CLPC1 trap covered a large part of the mature EXE2 protein sequence suggesting that this may have been the intact protein. Both EXE1 and EXE2 have UVR domains, similar as CLPF and CLPC1,2, suggesting that EXE1,2 are somehow functionally connected to the CLP system. Genetic analyses have shown that EXE1 and EXE2 are partially redundant in the 1O2 signaling pathway

68.

EXE1 is located in the thylakoid grana

margins and its function in 1O2 signaling requires its degradation by thylakoid FTSH2 protease 69, 79.

The primary function of EXE2 was suggested to be a modulator attenuating and controlling

the activity of EXE1 80. It seems possible that EXE2 functions as an adaptor or anti-adaptor for degradation products of thylakoid EXE1 that were generated by thylakoid FTSH2. In the adaptor scenario, the EXE1 fragments are part of the 1O2 retrograde signaling pathway and further degradation by the CLP system produces additional EXE1 cleavage products involved in the signaling pathway; here EXE2 would help deliver these EXE1 fragments to CLPC. In the antiadaptor scenario, EXE2 would interact with EXE1 and thereby partially protect EXE1 for degradation by FTHS2. Alternatively, CLP dependent degradation of EXE2 would produce


Page 25 of 40 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


degradation fragments that contribute to the retrograde signaling pathway. These various hypotheses should be tested in the future. The other proteins (#1, 2, 3, 4, 5, 13 and 16; Table 2) have no known functions, and have either no conserved domains or they have domains for which the functional significance is unknown (DUF760, DUF179). In particular proteins 1-5 are the most enriched proteins in the CLPC1-TRAP eluates, and they appear otherwise low abundant proteins only very infrequently detected in Arabidopsis leaf proteome or chloroplast samples (see PPDB). These proteins without known function should be considered candidate substrates or candidate CLPC adaptors; future studies should further determine their function and relationship to the chloroplast protease system. It should be noted that, with the exception of the CLPS adaptor, other Clp adaptors are not widely conserved (e.g. MecA), and they typically lack features that makes it obvious that they act as adaptors

6, 12

making their adaptor function difficult to prove. However, the presence of

chloroplast CLP adaptors could provide substrate selection specificity of the CLP system and it is therefore worthwhile testing for possible adaptor functions for these highly enriched proteins without known function. ACKNOWLEDGMENTS This research was supported by a grant from the National Science Foundation (NSF), Division of Molecular and Cellular Biosciences (MCB) #1614629 to K.J.V.W. C.M. was supported by a postdoctoral fellowship of the Swiss National Science Foundation # 159003. J-Y.R.L was supported by a three-year fellowship from the Ministry of Education (MOE) in Taiwan. We thank Qi Sun from Computational Biology Service Unit of Cornell University for his support with the PPDB and proteomics pipeline. We thank other members of the van Wijk lab for help and discussions. TABLES



Page 26 of 40

Table 1. CLP subunits identified by MS/MS in two independent CLPC-WT or CLPC1-TRAP affinity purifications (a). # MS/MS spectra (b) rep. 1 WT

rep. 2 WT

rep. 1 TRAP

rep. 2 TRAP

CLPF (adaptor)

0

6

0

34

CLPP1 (R-ring)

2

4

18

18

CLPP3 (P-ring)

0

0

13

9

CLPP4 (P-ring)

0

7

46

38

CLPP5 (P-ring)

3

5

45

38

CLPP6 (P-ring)

0

2

25

24

CLPR1 (R-ring)

0

1

10

12

CLPR2 (R-ring)

0

0

20

15

CLPR3 (R-ring)

0

2

31

18

CLPR4 (R-ring)

4

3

19

15

CLPT1 (with P-ring)

0

1

19

30

CLPT2 (with P-ring)

2

2

31

27

name

(a) Detailed information about the MS-based identification can be found in Table S1 (b) # MS/MS spectra corresponfd to the total number of observed adjSPC for these proteins detailed in Table S1.


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Table 2. Chloroplast proteins only found in eluates of CLPC1-TRAP but not in eluates of CLPC1-WT. Proteins identified with just a single peptide (even if observed multiple times) were discarded (^). #

accession

nam e

fam ily & dom ain

com m ent

function

adjSPC (repl. 1; 2) (h)

# unique peptides (h)

1

AT1G48450.1

unknow n protein (DUF760) #1 (TAIR annotation as alaninetRNA ligase - from UniPROT)

a

Only detected in CLPC1-TRAP and in clpr2-1 (4 exp.); not anyw here else in PPDB. Nearly complete intact protein (minus cTP)

unknow n

24; 48

21 total; 3 semitryptic

2

AT3G17800.1 unknow n protein (DUF760) #2

a

Very high CLPC1-TRAP; also detected in clpr2-1 (4 exp.)

unknow n

16; 23

13 tryptic

3

AT3G29240.1

unknow n function (DUF179)

b

Identified classical N-terminus C-SLS. With 2 different semi-tryptic peptides; each 2x). Enriched in clpr2-1 , clpc1 and clpc1 clps1

unknow n

24; 27

15 total; 4 semitryptic

4

AT1G63610.1

unknow n protein

c

significantly increased in clps1 and clpc1 [23]

unknow n

15; 24

11 total; no semitryptic

5

AT2G14910.1

unknow n protein

c

not observed previously in PPDB

unknow n

10; 11

10 tryptic

6

AT4G11570.1

ARPP phosphatase 2 (FHy2; PYRP2)

d

high in clpc1 clps1 and clpc1; once observed in clpr2-1. Not in w t plants

riboflavin synthesis

11; 36

17 tryptic; 2 semitryptic

7

AT3G10970.1

Haloacid hydrolase (HAD) *

d


unknow n

2; 18

11 tryptic

8

AT1G62180.1 AT4G04610.1 AT4G21990.1

5'-adenylylsulfate reductase1,2,3 (APR1,2,3)

e

Identified both the N-terminus and C-terminus. Family of three APR proteins (APR1,2,3). APR2 high in clpc1 clps1 [23]

Sassimilation

22;34


9

AT1G27510.1

EXECUTER 2 (EXE2)

f

observed in all three replicates of clpc1-1 and of signalling.R clpc1-1 clps1 , but not in w t [23]. Also in tw o OS replicates of clpr2-1 [54]

4; 12

7 tryptic

10

AT2G44650.1

CPN10-1

ATCG01240.1 11 ATCG00900.1

abundant; only C-terminal half detected

protein folding

8; 7

4 tryptic

30S ribosomal protein S7A,B

the tw o proteins are 100% identical

protein synthesis

5; 6

2 tryptic

12

AT3G25860.1

E2 - dihydrolipoamide acetyltransferase, PDH

highly abundant; observed in many proteomics papers and in PPDB. High in clpc1 clps1 [23]

lipid metabolism

5; 4

4 tryptic

13

AT5G52960.1

unknow n protein

CLPS1 interactor [23]. C-terminus detected. Phosphoproteomics studies suggest high phosphorylation.

unknow n

2; 4

3 tryptic

14

AT4G13430.1

isopropylmalate isomerase (IPMI)

highly abundant; observed in many proteomics papers and in PPDB

leucine synthesis

1; 3

2 tryptic

15

AT5G35790.1

glucose-6-phosphate 1dehydrogenase 1 (G6PD-1)

observed in multiple CLPS1 affinity preparations

OPP

1; 3


16

AT5G24060.1

unknow n protein *

g


unknow n

12

7 tryptic

17

AT4G33510.1

DAHP synthetase 2 (DHS2)

i

significantly increased in clpc1 and clpc1 clps1 and CLPS1 interactor [23]

shikimate pathw ay

10

5 tryptic

^

A complete list of proteins and their associated MS data can be found in Supporting Information - Table S1 and S2.

(a)

DUF760. Family of 5 proteins: AT1G48450 w ith cTP (Table 1A), AT3G17800 w ith cTP (Table 1A); AT1G32160 w ith cTP; AT3G07310 w ith mTP - not observed in PPDB); AT5G48590 w ith cTP - not observed in PPDB)

(b)

DUF179. Three family members all cTP. AT3G29240 (Table 1A); AT1G33780 - in CLPC1-WT & CLPC1 TRAP - ratio 1.3 (based on NadjSPC) and 0.9 (normalized to CLPC): and a few other genotypes in PPDB; AT3G19780 not observed in PPDB)

(c)

group of three related proteins w ithout PFAM. AT1G63610 (Table 1A); AT2G14910 (Table 1A); AT5G14970.1 (not observed in PPDB). All w ith cTP. No shared peptides betw een the tw o observed proteins

Member of the large halo-acid dehydrogenase (HAD) superfamily. AT4G11570 & AT3G10970 both in Table 2 form a small clade. Only AT4G11570 has ARPP (d) phosphatase activity. [Mitochondrial PYRP3 is AT4G25840 - not found in this study. FHY1/PYRP1 -AT1G79790 is also plastid - in Table 1 - ~3 fold more in CLPC1-WT than CLPC1-TRAP ] (e) Family of three APR proteins (APR1,2,3) - AT1G62180.1, AT4G04610.1 and AT4G21990.1 (f) UVR domain, similar as in EXE1, CLPF, CLPC1 and CLPC2 (g) Small family of three proteins: AT5G24060.1 (this Table), closely related AT3G49140.1 and the more distant AT3G59300.1 (not identified in PPDB) (h)

adjSPC - adjusted spectral counts. Table S3 provides information of all identified peptides for each of the proteins in this Table 2. Figure S5 show s sequence coverage for each protein in this Table 2.

(i) Small family of three DHAP synthases - AT4G33510.1 (This Table), AT1G22410.1 and AT4G39980.1



Page 28 of 40

FIGURE LEGENDS Figure 1. Generation of a chloroplast CLPC1 substrate trap in Arabidopsis. (A) Linear model of CLPC1 showing the chloroplast transit peptide (cTP), the N-domain (with repeats 1 and 2) involved in binding of adaptors (e.g. CLPS1) and substrates, the uvrB/C domain of unknown function, and two AAA+ domains (AAA1 and AAA2) involved in ATP depended unfolding of substrates. The AAA+ domains contain a conserved Walker B motif in which glutamate (E) residues are necessary for the hydrolysis of ATP. Sequence alignment of the AAA+ domains for Arabidopsis CLPC1, S. aureus CLPC (SaCLPC), E. coli CLPA (EcCLPA) and CLPB (EcCLPB) is shown, with the Walker B domains underlined. The critical glutamate residues are marked with an arrowhead and are mutated to an alanine in the TRAP constructs. (B) The CLPC1 substrate trap (CLPC1-TRAP) construct was generated by mutation of the glutamates of the two Walker B motifs of CLPC1 and addition of a STREPII tag to its C-terminus. A construct with the wt CLPC1 sequence and a C-terminal STREPII tag was generated as a control. The two constructs driven by a 35S promoter were stably transformed in both wt and the clpc1-1 null mutant resulting in three different heterozygous lines, wt/CLPC1-WT and clpc11/CLPC1-WT with no obvious phenotypes, and wt/CLPC1-TRAP which was clearly smaller and virescent. No clpc1-1/CLPC1-TRAP line could be recovered despite extensive efforts. The sequence of the STREPII tag is WSHPQFEK. Figure 2. Phenotypes of CLPC1-WT and CLPC1-TRAP lines (A) 4 weeks old T2 plants of the wt/CLPC1-WT, wt/CLPC1-TRAP and clpc1-1/CLPC1-WT lines. In the wt/CLPC1-TRAP line, three distinct phenotypes segregated, namely wt-like (wt*; aa), paler virescent smaller plants (Aa) and dwarf-albino plants (AA) were identified. The homozygous clpc1-1 and clpc2-2 null mutants

23

and wt are shown as controls. wt/CLPC1-WT and clpc1-

1/CLPC1-WT did not have visible phenotypes and were indistinguishable from wt. (B) Accumulation of recombinant CLPC1-WT or CLPC1-TRAP proteins in total soluble leaf protein extracts in the lines shown in (A) as determined by SDS-PAGE and immunoblotting using an anti-STREPII antibody. The lower panel shows the Ponceau stain of this blot to illustrate protein loading. Bands with the RIBULOSE CARBOXYLASE/OXYGENASE large subunit (RBCL) is indicated. The leaf extracts from the dwarf-albino (wt/CLPC1-TRAP (AA)) and pale clpc1-1 have visibly reduced levels of RUBISCO. 28 ACS Paragon Plus Environment

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Figure 3. Dominant negative effect of the CLPC1-TRAP construct. (A) Segregation analysis of the progenies of seven virescent T2 wt/CLPC1-TRAP lines (Aa) on agar plates without selective medium. We note that none of the albino-dwarf T2 individuals (AA) produced any seed. (B) Chi-square test of seven T2 parents shows that the virescent and albino phenotype of the wt/CLPC1-TRAP line segregate in a 2:1 ratio. 𝝌2 (2, N=125) = 0.984, P > 0.05. Figure 4. Immunoblot analysis of leaf soluble protein extracts and affinity eluates from homozygous wt/CLPC1-WT lines and heterozygous wt/CLPC1-TRAP lines. (A) After 13 days of growth on agar plants with ½ MS, 2% sucrose and 20 mg/L BAR as selectable marker for the transgenes, seedlings were transferred to soil and grown for 37 days under a 10/14 light/ dark at 100 µmol photons.m-2.s-1. The image shows an example of the two genotypes at the time of harvest. (B-E) Accumulation of CLPC1, CLPC2, CLPD, STREPII-tagged CLPC1, CLPR2, CLPP6, CLPS1, CLP and GLUTR in total soluble leaf extracts (B,C) or affinity eluates (D,E) of soil grown wt/CLPC1-WT and the heterozygous wt/CLPC1-TRAP as determined by SDS-PAGE and immunoblotting using specific antisera. Multiple gels were generated that were subsequently blotted to accommodate immune-detection with the specific antisera. The blots were reprobed with one or more sera. Panels B (total soluble leaf extract) and D (affinity eluate) show examples of Ponceau stains of blots as control of protein loading and molecular mass markers. ^ The titer of the antiserum against CLPD has a low titer and only very limited amounts are available. This low titer explains the lack of an increased response between 10 and 20 μgram (panel C). * For evaluation of the affinity eluates, gels were loaded with 1 and 10 μl of each eluate in case of CLPD and GLUTR, and 1 and 5 μl for the other sera. Figure 5. CLPC1-WT and CLPC1-TRAP protein interactors. The STREPII-tagged CLPC1-WT and CLPC1-TRAP proteins and their interactomes were affinity purified under non-denaturing conditions from soluble protein leaf extracts with two independent replicates each. Following SDS-PAGE, in-gel-tryptic digestion and MS/MS, proteins were annotated and quantified based on matched adjSPC and normalized to CLPC abundance (matched adjSPC to CLPC1,2) within each replicate. 29 ACS Paragon Plus Environment


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(A) Cross-correlation of protein abundance identified in eluates from CLPC1-WT and CLPC1TRAP based on relative abundance. Replicates were averaged. CLP subunits, endogenous biotin binding proteins that have high affinity for the streptactin column, and candidate CLPC1 substrates as listed in Table 2, are marked with different symbols and colors. (B) Relative abundance of CLP subunits in the CLPC1-TRAP and CLPC1-WT eluates that are part of the P-ring (CLPP3-6), R-ring (CLPP1, CLPR1-4) or CLPT1,2. This illustrates that the CLPPR core and associated CLPT1,T2 subunits are 6-9-fold enriched in the CLPC1-TRAP eluates compared to CLPC1-WT. Figure 6. The CLPC to CLP core interactions are stabilized in the CLPC1-TRAP line (A) Analysis by MS/MS of CLPC1-WT (left hand panel) and CLPC1-TRAP (right hand panel) eluates separated by native PAGE. Each gel lane was cut in nine slices, digested with trypsin and analyzed by MS/MS. Spectral counts (adjSPC) were calculated for each gel slice. Spectral counts for the large RUBISCO subunit (RBCL), for the CLPPRT subunits and for CLPC1,2 were summed and plotted for the CLPC1-WT plants and CLPC1-TRAP plants. Most of RBCL as well as RBCS elute as part of the 550 kDa RUBISCO holocomplex. (B) Immunoblot analysis of the CLPC1-WT and CLPC1-TRAP eluates separated by native PAGE using an anti-STREPII antibody. This shows that most of the STREPII-tagged CLPC1 runs as a ~200 kDa complex on the native PAGE gel. Arrows indicate higher molecular mass complexes containing CLPC1-TRAP protein. Figure 7. Working model of the CLP chloroplast proteolytic cycle and CLPC1 to CLP core interactions. The different steps include: i) priming of the chaperone by adaptors and/or ATP leading to formation of the activate hexamer in ATP-bound state, ii) recognition and interaction with substrates through the CLPC N-domain, possibly assisted by adaptors, iii) formation of the active CLPC-CLPPR core assembly, iv) unfolding of bound substrate by CLPC driven by ATP hydrolysis, and threading and delivery into the CLP protease chamber, v) cleavage of peptidyl bonds by CLP core catalytic activity concomitant with unfolding and threading by CLPC, vi) dissociation of CLPC chaperones and the CLPPR core complex and return to resting states. The following supporting information is available free of charge at ACS website http://pubs.acs.org 30 ACS Paragon Plus Environment

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SUPPORTING INFORMATION Table S1. Proteins identified in the affinity eluates of CLPC1-WT and CLPC1-TRAP based on tryptic and semi-tryptic searches. Table S2. Proteins only found in eluates of CLPC1-TRAP but not in eluates of CLPC1-WT. Table S3. Primers used in the study. Figure S1. Transcript levels of CLPC1, CLPC2, CLPC1-WT, CLPC1-TRAP in various genotypes Figure S2. Dominant negative effect of the CLPC1-TRAP protein. Figure S3. MS/MS peptides that demonstrate the Glu to Ala mutations (E374A and E718A) in CLPC1-TRAP protein as compared to the respective wild-type peptides. Figure S4. Protein sequence alignment of CLPC1 and CLPC2, and the peptide that provide experimental evidence for the mutations in the Walker B domains. Figure S5. Protein sequence alignment of CLPC1, CLPC2 and CLPD and peptides matching to CLPD identified by MS/MS. Figure S6. Protein sequence coverage by MS/MS of CLPF in the affinity eluates Figure S7. Primary sequences of proteins in Table 2 and their sequence coverage from MS/MS spectra from the affinity eluates. REFERENCES 1.

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In Vivo Trapping of Proteins Interacting with the Chloroplast CLPC1

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