Fahrner, M. | bioRxiv Science

bioRxiv2020

A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening/ Clearance and synergy of Orai1 gating checkpoints controls pore opening

The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints is required to allow STIM1 coupling and Orai1 pore opening. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/207183v2_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@3f80f1org.highwire.dtl.DTLVardef@12a6a0aorg.highwire.dtl.DTLVardef@197b825org.highwire.dtl.DTLVardef@1bab109_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Discovery of a novel small protein factor involved in the coordinated degradation of phycobilisomes in cyanobacteria

Phycobilisomes are the major pigment-protein antenna complexes that perform photosynthetic light harvesting in cyanobacteria, rhodophyte and glaucophyte algae. Up to 50% of the cellular nitrogen can be stored in their giant structures. Accordingly, upon nitrogen depletion, phycobilisomes are rapidly degraded. This degradation is tightly coordinated, follows a genetic program and involves small proteins serving as proteolysis adaptors. Here, we describe the role of NblD, a novel factor in this process in cyanobacteria. NblD is a cysteine-rich, 66-amino acid small protein that becomes rapidly induced upon nitrogen starvation. Deletion of the nblD gene in the cyanobacterium Synechocystis prevents the degradation of phycobilisomes, leading to a nonbleaching (nbl) phenotype. Competition experiments provided direct evidence for the physiological importance of NblD. Complementation by a plasmid-localized gene copy fully restored the phenotype of the wild type. Overexpression of NblD under nitrogen-replete conditions showed no effect, in contrast to the unrelated proteolysis adaptors NblA1 and NblA2, which can trigger phycobilisome degradation ectopically. Transcriptome analysis revealed that nitrogen starvation correctly induces nblA1/2 transcription in the {Delta}nblD strain implying that NblD does not act as a transcriptional (co-)regulator. However, fractionation and coimmunoprecipitation experiments indicated the presence of NblD in the phycobilisome fraction and identified the {beta}-phycocyanin subunit as its target. These data add NblD as a new factor to the genetically programmed response to nitrogen starvation and demonstrate that it plays a crucial role in the coordinated dismantling of phycobilisomes when nitrogen becomes limiting. Significance StatementDuring genome analysis, genes encoding small proteins are frequently neglected. Accordingly, small proteins have remained underinvestigated in all domains of life. Based on a previous systematic search for such genes, we present the functional analysis of the small protein NblD in a photosynthetic cyanobacterium. We show that NblD plays a crucial role during the coordinated dismantling of phycobilisome light-harvesting complexes. This disassembly is triggered when the cells run low in nitrogen, a condition that frequently occurs in nature. Similar to the NblA proteins that label phycobiliproteins for proteolysis, NblD binds to phycocyanin polypeptides but has a different function. The results show that, even in a well-investigated process, crucial new players can be discovered if small proteins are taken into consideration.

microbiology↗