Identification of downstream components of ubiquitin-conjugating enzyme PHOSPHATE2 by quantitative membrane proteomics in Arabidopsis roots

Abstract

MicroRNA399-mediated regulation of the ubiquitin-conjugating enzyme UBC24/phosphate2 (PHO2) is crucial for Pi acquisition and translocation in plants. Because of a potential role for PHO2 in protein degradation and its association with membranes, an iTRAQ (for isobaric tags for relative and absolute quantitation)- based quantitative membrane proteomic method was employed to search for components downstream of PHO2. A total of 7491 proteins were identified from Arabidopsis thaliana roots by mass spectrometry, 35.2% of which were predicted to contain at least one transmembrane helix. Among the quantifiable proteins, five were significantly differentially expressed between the wild type and pho2 mutant under two growth conditions. Using immunoblot analysis, we validated the upregulation of several members in phosphate transporter1 (PHT1) family 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 that they play roles as downstream components of PHO2. Moreover, we showed that PHO2 interacts with PHT1s in the postendoplasmic 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.

Figure 1.

PHO2 Is a Membrane-Associated Protein. Immunoblot analysis of PHO2 protein in the soluble (S) and membrane (M) fractions isolated from the roots of wild-type (WT), pho2, and two independent PHO2-overexpressing lines (35S:PHO2). Fructose-1,6-bisphosphatase (FBP) and H⁺-ATPase served as markers for soluble and membrane proteins, respectively. The protein staining of the blot is shown below the immunoblot.

Figure 2.

Analysis of 7491 Identified Proteins from Membrane Proteomics. (A) The left panel illustrates the distribution of subcellular localization of total identified proteins. Based on our criterion, a total of 3713 (49.6%) proteins containing at least one transmembrane helix or present in at least two databases or analyses are classified as membrane or membrane-associated proteins. A total of 1177 (15.7%) proteins are present only in one database or analysis. The number and percentage of annotated or predicted membrane proteins revealed by the analysis of GO category, TMHMM, SUBA, and posttranslational modification (PTM) associated with membrane anchoring are shown on the right. (B) The significantly enriched GO terms in the categories of molecular function and biological process. The cluster frequency and genome frequency indicate the proportion of identified proteins and total proteins annotated in the specific group versus the total annotated Arabidopsis proteins, respectively. The Bonferroni adjustment was applied to correct the P values.

Figure 3.

Loss of Function of PHF1 and PHT1;1 Alleviates the Pi Toxicity of pho2. (A) The shoot Pi concentrations of 12-d-old wild type (WT), pht1;1, phf1, pho2, pht1;1 pho2, and phf1 pho2 (n = 4). (B) PHT1;1 has a greater contribution than PHT1;2 or PHT1;3 to the Pi accumulation in pho2. The shoot Pi concentrations of 13-d-old wild-type, pht1;1, pht1;2, pht1;3, pho2, pht1;1 pho2, pht1;2 pho2, and pht1;3 pho2 seedlings grown under Pi-sufficient conditions (n = 3). (C) PHT1;8 and PHT1;9 do not contribute to the Pi accumulation in pho2. The shoot Pi concentration of 13-d-old wild-type, pht1;8, pht1;9, pho2, pht1;8 pho2, pht1;9 pho2, and pht1;8 pht1;9 pho2 seedlings grown in Pi-sufficient media (n = 4). Ten seedlings were pooled as one replicate. The different letters indicate the statistical significance of variations analyzed by Duncan’s multiple range test (P < 0.05), and error bars represent sd. FW, fresh weight.

Figure 4.

Increased Accumulation of PHF1 and PHT1 Proteins in the pho2 Mutant. (A) Immunoblot analysis of PHT1;1/2/3, PHT1;4, and PHF1 proteins in the roots of 12-d-old wild-type and pho2 plants grown under Pi-sufficient (+P) and -deficient (−P) conditions. The bottom panels show the protein staining on the blot. (B) Immunoblot analysis of PHT1;1/2/3, PHT1;4, and PHF1 proteins in the roots of Pi-starved wild-type and pho2 seedlings replenished with Pi over 72 h. The bands corresponding to PHT1;4 are indicated by an arrowhead. Actin is used as a loading control.

Figure 5.

PHO2 Negatively Regulates the Protein Level of PHF1 and PHT1s. (A) Immunoblot analysis of PHT1;1/2/3, PHT1;4, and PHF1 proteins in the roots of 18-d-old wild-type (WT) and pho2, pht1;1, pht1;1 pho2, pht1;2, and pht1;2 pho2 plants grown under Pi-sufficient conditions. The bottom panel shows the protein staining on the blot. (B) Quantitative RT-PCR analysis of PHT1;1, PHT1;2, PHT1;3, PHT1;4, and PHF1 mRNA in the roots of the wild type and mutants. Error bars represent se (n = 3). Data significantly different from the corresponding controls are marked by an asterisk (pho2 versus the wild type, pht1;1 pho2 versus pht1;1, or pht1;2 pho2 versus pht1;2, 0.01 < *P < 0.05, Student’s t test). CT, cycle threshold.

Figure 6.

PHO2 Facilitates the Degradation of PHT1 Proteins. (A) Time-course analysis of PHT1;1/2/3, PHT1;4, and PHF1 proteins in the roots of 12-d-old wild-type (WT) and pho2 seedlings subjected to the treatment with 200 µM CHX under Pi-sufficient conditions. Actin is used as a loading control. The bands corresponding to PHT1;4 are indicated by an arrowhead. One representative image out of 4 replicates is shown. (B) The relative remaining amount of PHT1;1/2/3 and PHT1;4 proteins upon CHX treatment was calculated from (A) and plotted on a semilog graph. The protein level was normalized with the corresponding actin controls. Error bars represent se (n = 4).

Figure 7.

Protein–Protein Interactions between PHO2 and PHT1s and between PHF1 and PHT1s. (A) to (C) BiFC analysis of PHF1-dependent interaction between PHO2 and PHT1;1 (A) or between PHO2 and PHT1;4 (B) in tobacco leaves. The PHF1 construct was coinfiltrated in all experiments except where indicated. BiFC analysis of the interaction between PHF1 and PHT1;1 or PHT1;4 in tobacco leaves (C). Coexpression of N-terminal yellow fluorescent protein (nYFP) or C-terminal yellow fluorescent protein (cYFP) with the corresponding PHT1s, PHO2^C748A, or PHF1 constructs was used as negative controls. Bars = 10 µm. (D) to (F) Split-ubiquitin Y2H analysis of the interaction between PHO2 and PHT1;4 (D), PHF1 and PHT1;1 (E), or PHF1 and PHT1;4 (F). Coexpression of NubI or NubG with PHT1;4 or PHF1 was used as positive and negative controls, respectively.

Figure 8.

PHO2-Mediated Ubiquitination of PHT1;1 at the EM. (A) and (B) Immunoblot analysis by anti-PHT1;1/2/3 or antiubiquitin antibody following immunoprecipitation (IP) of total membrane proteins (A) or enriched PM and EM proteins (B) with anti-PHT1;1/2/3 antibody. Membrane proteins were isolated from the roots of 12-d-old wild-type (WT), pho2, and pht1;1 seedlings grown under Pi-sufficient conditions. (C) Assessment of the enrichment of isolated PM and EM fractions used in (B) by immunoblot analysis. H⁺-ATPase and PHF1 served as markers for the PM and EM protein, respectively. (D) Analysis was performed similar to in (A), except 6-d-old seedlings were used. The quantification of PHT1;1/2/3 in the input samples and ubiquitinated proteins normalized with the corresponding PHT1;1/2/3 is indicated.

Figure 9.

The Regulatory Mechanism of Pi Uptake and Root-to-Shoot Pi Translocation through PHT1s and PHO1 by PHO2. PHO2 localizes to the ER and Golgi and suppresses Pi uptake and xylem loading of Pi by mediating the protein degradation of PHT1s and PHO1, which takes place in the outer cell layer and stele of roots, respectively. To simplify the illustration, these two activities are drawn in the same cell. The pathway indicated by the dashed line requires further evidence. See text for more details. MVB, multivesicular body.