Gene Replacement in Arabidopsis Reveals Manganese Transport as an Ancient Feature of Human, Plant and Cyanobacterial UPF0016 Proteins.

The protein family 0016 (UPF0016) is conserved through evolution, and the few members characterized share a function in Mn2+ transport. So far, little is known about the history of these proteins in Eukaryotes. In Arabidopsis thaliana five such proteins, comprising four different subcellular localizations including chloroplasts, have been described, whereas non-photosynthetic Eukaryotes have only one. We used a phylogenetic approach to classify the eukaryotic proteins into two subgroups and performed gene-replacement studies to investigate UPF0016 genes of various origins. Replaceability can be scored readily in the Arabidopsis UPF0016 transporter mutant pam71, which exhibits a functional deficiency in photosystem II. The N-terminal region of the Arabidopsis PAM71 was used to direct selected proteins to chloroplast membranes. Transgenic pam71 lines overexpressing the closest plant homolog (CMT1), human TMEM165 or cyanobacterial MNX successfully restored photosystem II efficiency, manganese binding to photosystem II complexes and consequently plant growth rate and biomass production. Thus AtCMT1, HsTMEM165, and SynMNX can operate in the thylakoid membrane and substitute for PAM71 in a non-native environment, indicating that the manganese transport function of UPF0016 proteins is an ancient feature of the family. We propose that the two chloroplast-localized UPF0016 proteins, CMT1 and PAM71, in plants originated from the cyanobacterial endosymbiont that gave rise to the organelle.

Homologous Proteins of the Manganese Transporter PAM71 Are Localized in the Golgi Apparatus and Endoplasmic Reticulum.

Chloroplast manganese transporter 1 (CMT1) and photosynthesis-affected mutant 71 (PAM71) are two membrane proteins that function sequentially to mediate the passage of manganese across the chloroplast envelope and the thylakoid membrane. CMT1 and PAM71 belong to a small five-member protein family in Arabidopsis thaliana. The other three, photosynthesis-affected mutant 71 like 3 (PML3), PML4 and PML5 are not predicted to reside in chloroplast membranes. In this study, the subcellular localization of PML3:GFP, PML4:GFP and PML5:GFP was determined using transient and stable expression assays. PML3:GFP localizes to the Golgi apparatus, whereas PML4:GFP and PML5:GFP are found in the endoplasmic reticulum. We also examined patterns of PML3, PML4 and PML5 promoter activity. Although the precise expression pattern of each promoter was unique, all three genes were expressed in the leaf vasculature and in roots. Greenhouse grown single mutants pml3, pml4, pml5 and the pml4/pml5 double mutant did not exhibit growth defects, however an inspection of the root growth revealed a difference between pml3 and the other genotypes, including wild-type, in 500 µM manganese growth conditions. Strikingly, overexpression of PML3 resulted in a stunted growth phenotype. Putative functions of PML3, PML4 and PML5 are discussed in light of what is known about PAM71 and CMT1.

The Plastid Envelope CHLOROPLAST MANGANESE TRANSPORTER1 Is Essential for Manganese Homeostasis in Arabidopsis.

The transition metal manganese (Mn) is indispensable for photoautotrophic growth since photosystem II (PSII) employs an inorganic Mn4CaO5 cluster for water splitting. Here, we show that the Arabidopsis membrane protein CHLOROPLAST MANGANESE TRANSPORTER1 (CMT1) is involved in chloroplast Mn homeostasis. CMT1 is the closest homolog of the previously characterized thylakoid Mn transporter PHOTOSYNTHESIS-AFFECTED MUTANT71 (PAM71). In contrast to PAM71, CMT1 resides at the chloroplast envelope and is ubiquitously expressed. Nonetheless, like PAM71, the expression of CMT1 can also alleviate the Mn-sensitive phenotype of yeast mutant Δpmr1. The cmt1 mutant is severely suppressed in growth, chloroplast ultrastructure, and PSII activity owing to a decrease in the amounts of pigments and thylakoid membrane proteins. The importance of CMT1 for chloroplast Mn homeostasis is demonstrated by the significant reduction in chloroplast Mn concentrations in cmt1-1, which exhibited reduced Mn binding in PSII complexes. Moreover, CMT1 expression is downregulated in Mn-surplus conditions. The pam71 cmt1-1double mutant resembles the cmt1-1 single mutant rather than pam71 in most respects. Taken together, our results suggest that CMT1 mediates Mn2+ uptake into the chloroplast stroma, and that CMT1 and PAM71 function sequentially in Mn delivery to PSII across the chloroplast envelope and the thylakoid membrane.

Plants contain small families of UPF0016 proteins including the PHOTOSYNTHESIS AFFECTED MUTANT71 transporter.

PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein that functions in manganese uptake into the lumen. Manganese is needed in the thylakoid lumen to build up the inorganic Mn4CaO5 cluster, the catalytic center for water oxidation, and is hence indispensable for oxygen evolution. A recent study revealed that PAM71 is well conserved in plants and shares homology to GCR1 DEPENDENT TRANSLATION FACTOR1 (GDT1) and TRANSMEMBRANE PROTEIN 165 (TMEM165) in Saccharomyces cerevisiae and Homo sapiens, respectively. In most eukaryotes only single members of this family, designated "Uncharacterized Protein Family 0016" (UPF0016), are present; however, plant genomes contain genes for several UPF0016 proteins. In Arabidopsis thaliana, this protein family comprises 5 members, which mainly differ in their N-terminal regions. PAM71 and its closest homolog PAM71-HL possess chloroplast transit peptides at their N-terminus. Two of the remaining 3 members are derived from a segmental chromosomal duplication event and lack an N-terminal extension. Thus, plants have evolved UPF0016 members residing in various compartments of the cell, whereas in non-plant eukaryotes just a Golgi localization occurs. The identification of PAM71 as a candidate Mn2+ transporter opens the question on the function of the remaining plant members. Here we resume briefly our current knowledge of UPF0016 members in Arabidopsis in comparison to their yeast and human UPF0016 members.

The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis.

In plants, algae, and cyanobacteria, photosystem II (PSII) catalyzes the light-driven oxidation of water. The oxygen-evolving complex of PSII is a Mn4CaO5 cluster embedded in a well-defined protein environment in the thylakoid membrane. However, transport of manganese and calcium into the thylakoid lumen remains poorly understood. Here, we show that Arabidopsis thaliana PHOTOSYNTHESIS AFFECTED MUTANT71 (PAM71) is an integral thylakoid membrane protein involved in Mn(2+) and Ca(2+) homeostasis in chloroplasts. This protein is required for normal operation of the oxygen-evolving complex (as evidenced by oxygen evolution rates) and for manganese incorporation. Manganese binding to PSII was severely reduced in pam71 thylakoids, particularly in PSII supercomplexes. In cation partitioning assays with intact chloroplasts, Mn(2+) and Ca(2+) ions were differently sequestered in pam71, with Ca(2+) enriched in pam71 thylakoids relative to the wild type. The changes in Ca(2+) homeostasis were accompanied by an increased contribution of the transmembrane electrical potential to the proton motive force across the thylakoid membrane. PSII activity in pam71 plants and the corresponding Chlamydomonas reinhardtii mutant cgld1 was restored by supplementation with Mn(2+), but not Ca(2+) Furthermore, PAM71 suppressed the Mn(2+)-sensitive phenotype of the yeast mutant Δpmr1 Therefore, PAM71 presumably functions in Mn(2+) uptake into thylakoids to ensure optimal PSII performance.