Background

Mn plays a vital role in the active sites of several enzymes such as the oxygen-evolving complex in photosystem II. In contrast to its role as an important micronutrient, Mn can be toxic when present in excess. It is therefore of crucial importance for cyanobacteria to maintain the intracellular levels of Mn and in particular to avoid free Mn in the cytoplasm. The cyanobacterium Synechocystis addresses this challenge by storing about 80% of the Mn in the periplasm. Only 20% of the cellular content can be detected in the cytoplasm and thylakoid system (Keren et al., 2002), with most of the Mn being incorporated into proteins, leaving virtually no free Mn in the cytoplasm. We recently identified the manganese transport protein Mnx, which resides in the thylakoid membrane and facilitates export of Mn from the cytoplasm into the thylakoid lumen in Synechocystis. According to our study, Mnx is a major player in maintaining the cellular Mn homeostasis (Brandenburg et al., 2017). To analyze the biological significance of Mnx, we developed a protocol to measure the periplasmic and intracellular Mn pools separately. We chose ICP-MS for quantification, since it is a sensitive and reliable method to detect metals in biological samples. Detection limits can be in the range of [ng L^-1] and below. Prior to the analysis, all complex molecules of the sample are broken down to atomic compounds by digestion with nitric acid. Subsequently, the sample is ionized by an inductively coupled plasma and analyzed by mass spectrometry. In this protocol, we describe the detailed workflow for subcellular Mn quantification, from sampling to the calculation of Mn concentrations.