Functional and Developmental Impact of Cytosolic Protein N-Terminal Methionine Excision in Arabidopsis

Protein N-terminal methionine (Met) excision (NME) is carried out by two types of Met aminopeptidases (MAPs), MAP1 and MAP2, in eukaryotes. Three enzymes, MAP1A, MAP2A, and MAP2B, have been identified in the cytoplasm of Arabidopsis (Arabidopsis thaliana). MAP transcript quantification revealed a predominance of MAP2B and developmental and organ-specific regulation of both MAP1A and MAP2s. By combining reverse genetics and reverse chemogenomics in transgenic plant lines, we have devised specific and reversible switches for the investigation of the role of cytoplasmic NME in Arabidopsis and of the respective contributions of the two types of cytoplasmic MAPs throughout development. dsRNA interference and knockout (KO) plant lines targeting either MAP1A alone or both MAP2s simultaneously were constructed and shown to display wild-type phenotypes. In the MAP1A KO context, modulating MAP2 activity by treatment with various concentrations of the specific drug fumagillin impaired plant development, with particularly strong effects on the root system. Reciprocally, complete MAP2 inhibition in various MAP1A knocked-down genetic backgrounds also generated a gradient of developmentally abnormal plants, but the effects on the root system were milder than in the KO context. In the absence of MAP2 activity, the severity of the phenotype in the MAP1A knocked-down lines was correlated to the extent of MAP1A mRNA accumulation. Complete cytoplasmic NME inactivation blocked development after plant germination. Thus, in plants, (1) cytoplasmic NME is essential; (2) MAP1A and MAP2s are functionally interchangeable, which is not the case in fungi and animals, as a complete block of either MAP-type activity does not cause any visible molecular or phenotypic effect; and (3) a minimal level of cytoplasmic MAP is required for normal development

(15)N-Metabolic labeling for comparative plasma membrane proteomics in Arabidopsis cells.

An important goal for proteomic studies is the global comparison of proteomes from different genotypes, tissues, or physiological conditions. This has so far been mostly achieved by densitometric comparison of spot intensities after protein separation by 2-DE. However, the physicochemical properties of membrane proteins preclude the use of 2-DE. Here, we describe the use of in vivo labeling by the stable isotope (15)N as an alternative approach for comparative membrane proteomic studies in plant cells. We confirm that (15)N-metabolic labeling of proteins is possible and efficient in Arabidopsis suspension cells. Quantification of (14)N versus (15)N MS signals reflects the relative abundance of (14)N and (15)N proteins in the sample analyzed. We describe the use of (15)N-metabolic labeling to perform a partial comparative analysis of Arabidopsis cells following cadmium exposure. By focusing our attention on plasma membrane proteins, we were able to confidently identify proteins showing up to 5-fold regulation compared to unexposed cells. This study provides a proof of principle that (15)N-metabolic labeling is a useful technique for comparative membrane proteome studies

Phosphatidylinositol 3-phosphate-binding protein AtPH1 controls the localization of the metal transporter NRAMP1 in Arabidopsis.

"Too much of a good thing" perfectly describes the dilemma that living organisms face with metals. The tight control of metal homeostasis in cells depends on the trafficking of metal transporters between membranes of different compartments. However, the mechanisms regulating the location of transport proteins are still largely unknown. Developing Arabidopsis thaliana seedlings require the natural resistance-associated macrophage proteins (NRAMP3 and NRAMP4) transporters to remobilize iron from seed vacuolar stores and thereby acquire photosynthetic competence. Here, we report that mutations in the pleckstrin homology (PH) domain-containing protein AtPH1 rescue the iron-deficient phenotype of nramp3nramp4 Our results indicate that AtPH1 binds phosphatidylinositol 3-phosphate (PI3P) in vivo and acts in the late endosome compartment. We further show that loss of AtPH1 function leads to the mislocalization of the metal uptake transporter NRAMP1 to the vacuole, providing a rationale for the reversion of nramp3nramp4 phenotypes. This work identifies a PH domain protein as a regulator of plant metal transporter localization, providing evidence that PH domain proteins may be effectors of PI3P for protein sorting

Extent of N-terminal modifications in cytosolic proteins from eukaryotes.

International audienceMost proteins in all organisms undergo crucial N-terminal modifications involving N-terminal methionine excision, N-alpha-acetylation or N-myristoylation (N-Myr), or S-palmitoylation. We investigated the occurrence of these poorly annotated but essential modifications in proteomes, focusing on eukaryotes. Experimental data for the N-terminal sequences of animal, fungi, and archaeal proteins, were used to build dedicated predictive modules in a new software. In vitro N-Myr experiments were performed with both plant and animal N-myristoyltransferases, for accurate prediction of the modification. N-terminal modifications from the fully sequenced genome of Arabidopsis thaliana were determined by MS. We identified 105 new modified protein N-termini, which were used to check the accuracy of predictive data. An accuracy of more than 95% was achieved, demonstrating (i) overall conservation of the specificity of the modification machinery in higher eukaryotes and (ii) robustness of the prediction tool. Predictions were made for various proteomes. Proteins that had undergone both N-terminal methionine (Met) cleavage and N-acetylation were found to be strongly overrepresented among the most abundant proteins, in contrast to those retaining their genuine unblocked Met. Here we propose that the nature of the second residue of an ORF is a key marker of the abundance of the mature protein in eukaryotes

Proton exchange by the vacuolar nitrate transporter CLCa is required for plant growth and nitrogen use efficiency

International audienceNitrate is a major nutrient and osmoticum for plants. To deal with fluctuating nitrate availability in soils, plants store this nutrient in their vacuoles. Chloride channel a (CLCa), a 2NO À 3 /1H + exchanger localized to the vacuole in Arabidopsis (Arabidopsis thaliana), ensures this storage process. CLCa belongs to the CLC family, which includes anion/proton exchangers and anion channels. A mutation in a glutamate residue conserved across CLC exchangers is likely responsible for the conversion of exchangers to channels. Here, we show that CLCa with a mutation in glutamate 203 (E203) behaves as an anion channel in its native membrane. We introduced the CLCa E203A point mutation to investigate its physiological importance into the Arabidopsis clca knockout mutant. These CLCa E203A mutants displayed a growth deficit linked to the disruption of water homeostasis. Additionally, CLCa E203A expression failed to complement the defect in nitrate accumulation of clca and favored higher N-assimilation at the vegetative stage. Further analyses at the post-flowering stages indicated that CLCa E203A expression results in an increase in N uptake allocation to seeds, leading to a higher nitrogen use efficiency compared to the wild-type. Altogether, these results point to the critical function of the CLCa exchanger on the vacuole for plant metabolism and development