Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas

Abstract

MscS is a mechanosensitive channel that is ubiquitous among bacteria. Recent progress in the genome projects has revealed that homologs of MscS are also present in eukaryotes, but whether they operate as ion channels is unknown. In this study we cloned MSC1, a homolog of MscS in Chlamydomonas, and examined its function when expressed in Escherichia coli. Full-length MSC1 was not functional when expressed in E. coli cells. However, removal of the N-terminal signal sequence (⌬N-MSC1) reversed this effect. ⌬N-MSC1 was found to open in response to membrane stretch and displayed a preference for anions over cations as permeable ions. ⌬N-MSC1 exhibited marked hysteretic behavior in response to ascending and descending stimuli. That is, channel gating occurred in response to significant stimuli but remained open until the stimulus was almost completely removed. Indirect immunofluorescence revealed that MSC1 is present as punctate spots in the cytoplasm and chloroplasts. Moreover, knockdown of MSC1 expression resulted in the abnormal localization of chlorophyll. These findings show that MSC1 is an intracellular mechanosensitive channel and is responsible for the organization of chloroplast in Chlamydomonas.MscS ͉ hysteresis ͉ heterogeneous expression ͉ knockdown ͉ green algae M echanosensation is typically involved in auditory perception, touch sensation, proprioception, and gravity perception. The mechanoreceptor potential in sensory cells is mediated by mechanosensitive channels, which are activated by stretching or deformation of the membrane. The patch-clamp technique has revealed that mechanosensitive channels are present in almost every cell type, not just sensory cells. In fact, mechanosensitive channels were first recorded with the patch-clamp method in the muscle cell (1). Mechanosensitive channels in nonsensory cells are believed to be responsible for monitoring cell deformation evoked by contact with surrounding materials and by changes in osmolarity.Bacteria harbor the mechanosensitive channels MscS and MscL, which act to protect against hypoosmotic downshock (2). The presence of MscS homologs in virtually all eubacteria and archaea suggests that MscS has an essential function in prokaryotes, but whether every MscS homolog serves as a mechanosensitive channel is uncertain. For instance, although there are at least six MscS homologs in the Escherichia coli genome, only two of them (MscS and MscK) have been detected by electrophysiological methods (3, 4). Recent genomic projects on eukaryotes have uncovered the presence of MscS homologs in some plants (Arabidopsis and Oryza), protists (Chlamydomonas), and fungi (Schizosaccharomyces). Curiously, MscS homologs have not been found in animals (5, 6). The MscS homologs of Arabidopsis (MSL2 and MSL3) have been observed as one or two foci on the plastid envelope, and their double knockout results in an abnormal size and shape of the plastids (6). This finding suggests that one of the possible functions of the MscS homologs is to control the size and shape of the intr...

Conditional Depletion of the Chlamydomonas Chloroplast ClpP Protease Activates Nuclear Genes Involved in Autophagy and Plastid Protein Quality Control

“…Among the most highly upregulated transcripts were those of several small HSP proteins and chaperones, proteases, proteins involved in autophagy and thylakoid membrane biogenesis, protein kinases and transporters, and ion channels (Supplemental Data Set 4B). Among the latter, one gene of particular interest is Msc1, which encodes a mechanosensitive ion channel suggested to be localized both in the cytosol and chloroplast (Nakayama et al, 2007). It could be involved in sensing osmotic stress, allowing the cell to counteract changes in osmotic pressure during the cellular swelling that accompanies autophagy.…”

Organellar mechanosensitive channels in fission yeast regulate the hypo-osmotic shock response

“…The function of the chloroplast MscS-like proteins is to regulate chloroplast shape 10 and to protect plastids from hypo-osmotic stress during normal plant growth 36 . In Chlamydomonas, MSC1 is present as punctuate spots in the chloroplast and cytoplasm and involved in chlorophyll localization 11 . Thus, our study adds a new aspect of the MscS family in terms of its localization and function.…”

“…While MscS is required for the osmotic response in bacteria 6,8,9 , it is unknown whether eukaryotic MscS homologues are involved in the osmotic response. Rather, eukaryotic MscS homologues analyzed so far, such as Arabidopsis thaliana MscS-like proteins MSL2 and MSL3 and Chlamydomonas reinhardtii MscS homologue MSC1, are associated with chloroplast organization 10,11 . Interestingly, MSL2, MSL3 and MSC1 are expressed in organellar membranes, including the chloroplast.…”

The Ectocarpus genome and the independent evolution of multicellularity in brown algae

“…These include several channels that are likely to be involved in calcium signalling such as an inositol triphosphate/ ryanodine type receptor (IP3R/RyR), four 4-domain voltage-gated calcium channels, and an expanded family of 18 transient receptor potential channels. Members of all these classes are found in animal genomes but are absent from the genomes of land plants 21,22 . No IP3R genes have been identified in the sequenced diatom and oomycete genomes, but the presence of an IP3R in Ectocarpus is consistent with the demonstration of 'animal-like' fast calcium waves and inositolphosphate-induced calcium release in embryos of the brown alga Fucus serratus 23,24 .…”