# AnaToc75 (Alr2269) mediates calcium uptake across the outer membrane in Anabaena sp. PCC 7120

## TL;DR

This study shows that a cyanobacterial protein, Alr2269, functions as a calcium channel in the outer membrane, especially under low calcium conditions.

## Contribution

The study reveals a new role for a Toc75 homolog in cyanobacteria as a calcium channel, challenging previous assumptions about its function.

## Key findings

- Alr2269 is a specific channel for calcium uptake across the outer membrane in low-Ca2+ environments.
- Inactivation of Alr2269 reduces growth under Ca2+-limiting conditions but not under low-Mg2+ conditions.
- The protein is not essential for β-barrel protein insertion but is crucial for calcium transport.

## Abstract

It has been a long-standing notion that cyanobacterial Omp85 proteins are essential because of their role in insertion of β-barrel proteins into the outer membrane (OM). Alr2269 from Anabaena sp. PCC 7120 is such an Omp85 protein, structurally related to the Toc75 channel of the translocon at the outer envelope membrane of chloroplasts but is actually non-essential to Anabaena under calcium-replete conditions. In a completely segregated alr2269-null mutant, a predicted S-layer protein, All3983, and many β-barrel proteins in the OM were upregulated compared with the wild type. By removal of each component from the medium, Ca2+ deficiency was identified to be the key environmental signal for upregulation of Pall3983-luxAB in the mutant. Assays of 45Ca2+ uptake in the wild type and the mutant indicated that Alr2269 is a relatively specific channel for transport of Ca2+ across the OM in low-Ca2+ medium. Accordingly, inactivation of alr2269 greatly reduced the growth under Ca2+-limiting conditions, while the complemented strain grew as the wild type. In low-Mg2+ medium, however, the alr2269 mutant showed only a slight difference in growth from the wild type. These results establish Alr2269 (AnaToc75) as the main channel for Ca2+ uptake across the OM of Anabaena in low-Ca2+ environments.

Ca2+ is required for photosynthesis and various cellular activities in cyanobacteria, but Ca2+ uptake, in particular how Ca2+ is transported across the outer membrane, has been barely investigated in cyanobacterial species. In this study, we found that a cyanobacterial Toc75 homolog is not necessarily essential for protein integration into the outer membrane as was thought before but instead is required for Ca2+ uptake in low-Ca2+ environments. This finding not only establishes a Ca2+ channel across the outer membrane of cyanobacteria but also provides an example of additional functions for Omp85 proteins.