Investigating the Uptake of Arsenate by Chlamydomonas reinhardtii Cells and its Effect on their Lipid Profile using Single Cell ICP-MS and Easy Ambient Sonic-Spray Ionization-MS.

The complementary use of single cell atomic mass spectrometry (MS) and ambient molecular MS allowed for the in-depth study of arsenate uptake by Chlamydomonas reinhardtii cells and of the effect this toxic metalloid species has on their lipid profile. Compared to conventional inductively coupled plasma mass spectrometry (ICP-MS) analysis, in which case hundreds of thousands of cells are digested and then analyzed, it is demonstrated that single cell (SC) ICP-MS provides uptake data that are potentially of greater biological relevance. This includes the arsenic mass distribution within the cell population, which fits to a log-normal probability function, the most frequently contained arsenic mass within the cells (1.5-1.8 fg As per cell), and the mean arsenic uptake value (ranging from 2.7 to 4.1 fg As per cell for the three arsenate incubation concentrations, that is, 15, 22.5, and 30 µg As per mL) derived from the log-normal arsenic mass distribution within the cell population. The SC approach also allows for differentiating the arsenic present in and/or adsorbed on the cells, from the arsenic present in the extracellular solution, in a single analysis. In a similar fashion, ambient molecular MS in the form of desorption easy ambient sonic spray ionization (EASI) -MS was used to rapidly profile cell membrane lipids from cells spotted directly on a glass slide. EASI-MS analysis revealed that cells grown in the presence of increasing concentrations of arsenate exhibited changes in the degree of saturation of their membrane lipids, as was observed by the increasing intensity ratio of lipids with less unsaturated acyl chains to the same type of lipids with more unsaturated fatty acid chains. Thus, indicating "homeoviscous adaptation" of extraplastidial and thylakoid cell membranes, induced by the presence of arsenate.

N-terminus of the photosystem II manganese stabilizing protein: effects of sequence elongation and truncation.

The importance of the N-terminal domain of manganese stabilizing protein in binding to photosystem II has been previously demonstrated [Eaton-Rye and Murata (1989) Biochim. Biophys. Acta 977, 219-226; Odom and Bricker (1992) Biochemistry 31, 5616-5620]. In this paper, we report results from a systematic study of functional and structural consequences of N-terminal elongation and truncation of manganese stabilizing protein. Precursor manganese stabilizing protein is the unprocessed wild-type protein, which carries an N-terminal extension of 84 amino acids in the form of its chloroplastic signal peptide. Despite its increased size, this protein is able to reconstitute O(2) evolution activity to levels observed with the mature, processed protein, but it also binds nonspecifically to PSII. Truncation of wild-type manganese stabilizing protein by site-directed mutagenesis to remove three N-terminal amino acids, resulting in a mutant called DeltaG3M, causes no loss of activity reconstitution, but this protein also exhibits nonspecific binding. Further truncation of the wild-type protein by ten N-terminal amino acids, producing DeltaE10M, limits binding of manganese stabilizing protein to 1 mol/mol of photosystem II and decreases activity reconstitution to about 65% of that obtained with the wild-type protein. Because two copies of wild type normally bind to photosystem II, amino acids in the domain (4)K-(10)E must be involved in the binding of one copy of manganese stabilizing protein to photosystem II. Spectroscopic analysis (CD and UV spectra) reveals that N-terminal elongation and deletion of manganese stabilizing protein influence its overall conformation, even though secondary structure content is not perturbed. Our data suggest that the solution structure of manganese stabilizing protein attains a more compact solution structure upon removal of N-terminal amino acids.