Table 1. Identification of the Translocation Intermediate-Associating Proteins by LC-MS/MS
IdentifierTotal Spectrum Counts
Purified with Translocating pFd-TEV-Protein A from Chloroplasts Using IgG-SepharosePurified with Translocating pFdFLAG-TEV-Protein A from Chloroplast Membranes Using αFLAG-AgaroseAtChloro Databased
Mocka+ATPPostbRankcMocka+ATPPostbRankc
TOC components (sum):(0)(191)(25)(0)(251)(3)
 Toc75-III07513[2]0853[3]296
 Toc1590605[4]01040[2]389
 Toc340303[8]0340[10]67
 Toc330264[9]0280[12]64
TIC components (sum):(0)(233)(7)(4)(281)(6)
 Tic214(Ycf1)01574[1]42076[1]317
 Tic560453[5]0380[6]64
 Tic1000310[7]0360[8]51
 (Tic20-I)e0000000
Ycf2/FtsHi components (sum):(0)(137)(7)(0)(246)(0)
 Ycf20333[6]0670[4]50
 FtsH120220[10]0280[15]112
 FtsHi50200[11]0340[11]36
 NAD-MDH0174[13]0240[14]313
 FtsHi20160[15]0350[9]59
 FtsHi40160[16]0270[13]52
 FtsHi10130[18]0360[7]78
Other proteins commonly found in the specifically associating Top 20 protein lists of the both experiments
 Tgd4-like (At2g44640)0490[3]0550[5]55
 OEP160150[17]0170[20]198
 EMB2737 (At5g53860)090[20]0200[17]15
Other chaperones
 Hsp93 (-III and -V)6292001702411
 Cpn60α (At2g28000)1010230001195
 Hsp70 (At4g24280)9917030818
 Cpn60β (At1g55490)88270001427
 Hsp90C (At2g04030)000000331
Historical TIC candidate proteins
 Tic1100012000982
 Tic40000000215
 Tic55004030268
 Tic62031100089
 Tic3200000012
  • pFd-TEV-Protein A and pFdFLAG-TEV-Protein A were used for in vitro import experiments with Arabidopsis chloroplasts in the presence of 0.1 mM ATP. Translocation intermediates were purified either from the chloroplasts or from the membrane fraction of the chloroplasts using either the IgG-Sepharose or the αFLAG-agarose and analyzed by LC-MS/MS (+ATP).

  • a Mock-purified samples from the same amounts of chloroplasts or chloroplast membranes without the addition of pFd(FLAG)-TEV-Protein A were also analyzed as one negative control (Mock).

  • b Another negative control purification with the excess amount of pFd(FLAG)-TEV-Protein A but added only after solubilization of chloroplasts or chloroplast membranes was carried out and analyzed as well (Post).

  • c Only those proteins identified in the Top 20 protein lists (Rank) of the two independent experiments (+ATP) in common with enrichment index above 4 compared to both two different negative controls (Mock and Post) are displayed. All the TOC, TIC, and Ycf2 complex components except Tic20-I were retained in both Top 20 lists.

  • d For reference of their relative easiness/difficulty for MS identification in chloroplasts, total spectrum counts extracted from the AtChloro database (as of March 18, 2018) are shown.

  • e Note that Arabidopsis Tic20-I protein, a central component of TIC, has been known to be an extremely difficult membrane protein to be detected by MS, while this protein could be clearly detected as a translocation intermediate-associating protein by immunoblotting (Kikuchi et al., 2013).