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Conserved function of UPF0016 manganese transporter across human, plant and bacteria

ABSTRACT: The Uncharacterized Protein Family 0016 (UPF0016) is conserved throughout evolution. In humans, the member TMEM165 is a putative Golgi localized Ca2+ and/or Mn2+ transporter. Genes encoding UPF0016 proteins are also present in many bacteria, including the cyanobacteria Synechocystis (MNX/SynPAM71), where it has been described as Mn2+ exporter. In Arabidopsis, two members of this family are embedded in the chloroplast membranes; the CMT1 protein is localized in the inner envelope membrane and the PAM71 protein is localized in the thylakoid membrane, and both cooperate to transport Mn2+ to the oxygen evolving complex of photosystem II. We used the pam71 mutant of Arabidopsis as a platform to perform intra- and inter-species complementation assays. The N-terminus of PAM71 was fused in front of TMEM165, CMT1 and MNX to direct them into chloroplast membranes. We performed LC-MS/MS experiments to assess their sub-plastidial localization.

INSTRUMENT(S): impact II

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Giada Marino

LAB HEAD: Anja Schneider

PROVIDER: PXD022763 | Pride | 2021-04-20

REPOSITORIES: Pride

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Project description:Chloroplast is a major plant cell organelle that fulfills essential metabolic and biosynthetic functions. Located at the interface between the chloroplast and other cell compartments, the chloroplast envelope system is a strategic barrier controlling exchanges of ions, metabolites and proteins, thus regulating essential metabolic functions (synthesis of hormones precursors, amino acids, pigments, sugars, vitamins, lipids, nucleotides…) of the plant cell. However, chloroplast envelope membranes remain the hidden part of the chloroplast proteome and many envelope proteins remain to be characterized (known but uncharacterized envelope proteins) or identified (orphan known envelope-associated functions carried by unidentified proteins) Indeed, the envelope contains only 1% of the chloroplast proteins (i.e. 0.25% of the whole cell proteome). When comparing the composition crude leaf extract and purified envelope vesicles, a high theoretical enrichment factor (EF) of specific envelope proteins is thus expected. This is especially true for minor envelope proteins (e.g. representing 1/100 or 1/1000 of the envelope protein content). Sensitivity of MS technique has been greatly improved during the last decade. Today, thanks to the continuous improvement of MS techniques, we are able to detect more components in a complex sample, to generate quantitative data and to statistically validate above-cited enrichment factor. Here, we have taken advantage of present-days better MS sensitivity towards a better definition (differentiate genuine envelope proteins from contaminants) of the chloroplast envelope proteome. This MS- and statistical-based analysis relied on an enrichment factor calculated for each protein identified in purified envelope fractions when compared to the value obtained for the same protein in crude cell extracts. Using this approach, a total of 1376 proteins was detected in purified envelope fractions, of which, more than 500 could be assigned an envelope localization combining MS-based statistical analyses and manual annotation using data from the literature or prediction tools. Interestingly, many of such proteins being unknown or unexpected envelope components, these data constitute a new resource of significant value to the broader plant science community aiming to define principles and molecular mechanisms controlling fundamental aspects of plastid biogenesis and functions.

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Conserved function of UPF0016 manganese transporter across human, plant and bacteria

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