ABSTRACT: MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/PHO2 is crucial for phosphate (Pi) acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ-based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis roots by mass spectrometry, 35.2 % of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, 5 were significantly differentially expressed between the wild-type and pho2 mutant under 2 growth conditions. Using immunoblot analysis, we validated the upregulation of several PHT1 Pi transporters and PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) in pho2 and demonstrated that PHO2 mediates the degradation of PHT1 proteins. Genetic evidence that loss of PHF1 or PHT1;1 alleviated Pi toxicity in pho2 further suggests their roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the post-endoplasmic reticulum compartments and mediates the ubiquitination of endomembrane-localized PHT1;1. This study not only uncovers a mechanism by which PHO2 modulates Pi acquisition by regulating the abundance of PHT1s in the secretory pathway destined for plasma membranes, but also provides a database of the membrane proteome that will be widely applicable in root biology research. Spectra Processing: The MS/MS raw spectra generated from the Waters Q-TOF Premier (the first replicate) and the AB SCIEX TripleTOF 5600 (the second and third replicates) instruments were processed to detect MS/MS peaks using UniQua (Chang et al., 2013). For the UniQua processing, the spectra were converted into mzXML format using massWolf (version 4.3.1) and ProteoWizard (version 3.0.4623) for the raw data generated from the Waters and AB SCIEX instruments, respectively. The UniQua parameters for Waters Q-TOF Premier were: smoothing = 9, centroiding high = 80%, maximum resolution = 16,000, baseline cutoff = 1.5 counts, reporter m/z range = 114-117, and reporter dynamic range = 5-1,000. The UniQua parameters for AB SCIEX TripleTOF 5600 were: smoothing = 11, centroiding high = 80%, maximum resolution = 25,000, baseline cutoff = 4 counts, reporter m/z range= 114-117 and reporter dynamic range = 5-100,000. The processed MS/MS spectra were then converted into Mascot generic format (.mgf) using mzXML2Search in Trans Proteomics Pipeline (TPP)1 (Deutsch et al., 2010) version 4.4 rev. 1. To obtain better quantification results, the MS/MS spectra with peak number less than 10, average intensity of the top 10 abundant peaks less than 15 counts and iTRAQ average intensity less than 30 counts were further removed from the peaklist file. Protein Qualification and Quantitation: The processed spectra were searched against the TAIR10 database (December 2011, 27,416 sequences) using the Mascot version 2.3 (Matrix Science, London, UK). For the MASCOT search, the mass tolerance for peptide precursor and MS/MS fragments was 0.1 Da. Only one missed cleavage was allowed for tryptic peptides. The methylthiolation (C), iTRAQ4plex (N-term) and iTRAQ4plex (K) were set as fixed modification. The Oxidation (M) and iTRAQ4plex (Y) were set as variable modification. The quantitation option was enabled and set to the iTRAQ4-plex approach. The peptide was considered to be identified if the MASCOT ion score greater than 27 (P less than 0.05). In addition, the protein was considered to be identified if the MASCOT protein score was greater than or equal to 27 with at least one unique and two different peptides identified. For the protein quantitation, only unique peptide was quantified in MASCOT and used to determine the protein ratio in MASCOT. To minimize the systematic ratio error due to sample preparation, the protein ratios were normalized by the mode of the overall protein ratios. For the data acquired by the Q-TOF Premier, 72,817 peptides and 3107 proteins were quantified. For the data acquired by the TripleTOF 5600, 41,885 peptides and 2893 proteins were quantified in the first replicate; 82,397 peptides and 4939 proteins were quantified in the second replicate.
Chia-Li Han 
