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.