RESUMEN
OSCA/TMEM63 channels are the largest known family of mechanosensitive channels1-3, playing critical roles in plant4-7 and mammalian8,9 mechanotransduction. Here we determined 44 cryogenic electron microscopy structures of OSCA/TMEM63 channels in different environments to investigate the molecular basis of OSCA/TMEM63 channel mechanosensitivity. In nanodiscs, we mimicked increased membrane tension and observed a dilated pore with membrane access in one of the OSCA1.2 subunits. In liposomes, we captured the fully open structure of OSCA1.2 in the inside-in orientation, in which the pore shows a large lateral opening to the membrane. Unusually for ion channels, structural, functional and computational evidence supports the existence of a 'proteo-lipidic pore' in which lipids act as a wall of the ion permeation pathway. In the less tension-sensitive homologue OSCA3.1, we identified an 'interlocking' lipid tightly bound in the central cleft, keeping the channel closed. Mutation of the lipid-coordinating residues induced OSCA3.1 activation, revealing a conserved open conformation of OSCA channels. Our structures provide a global picture of the OSCA channel gating cycle, uncover the importance of bound lipids and show that each subunit can open independently. This expands both our understanding of channel-mediated mechanotransduction and channel pore formation, with important mechanistic implications for the TMEM16 and TMC protein families.
Asunto(s)
Canales de Calcio , Microscopía por Crioelectrón , Activación del Canal Iónico , Mecanotransducción Celular , Humanos , Anoctaminas/química , Anoctaminas/metabolismo , Canales de Calcio/química , Canales de Calcio/metabolismo , Canales de Calcio/ultraestructura , Lípidos/química , Liposomas/metabolismo , Liposomas/química , Modelos Moleculares , Nanoestructuras/químicaRESUMEN
Sweet potato [Ipomoea batatas (L.) Lam.] is an important food and industrial crop. Its storage root is rich in starch, which is present in the form of granules and represents the principal storage carbohydrate in plants. Starch content is an important trait of sweet potato controlling the quality and yield of industrial products. Vacuolar invertase encoding gene Ibßfruct2 was supposed to be a key regulator of starch content in sweet potato, but its function and regulation were unclear. In this study, three Ibßfruct2 gene members were detected. Their promoters displayed differences in sequence, activity, and cis-regulatory elements and might interact with different transcription factors, indicating that the three Ibßfruct2 family members are governed by different regulatory mechanisms at the transcription level. Among them, we found that only Ibßfruct2-1 show a high expression level and promoter activity, and encodes a protein with invertase activity, and the conserved domains and three conserved motifs NDPNG, RDP, and WEC are critical to this activity. Only two and six amino acid residue variations were detected in sequences of proteins encoded by Ibßfruct2-2 and Ibßfruct2-3, respectively, compared with Ibßfruct2-1; although not within key motifs, these variations affected protein structure and affinities for the catalytic substrate, resulting in functional deficiency and low activity. Heterologous expression of Ibßfruct2-1 in Arabidopsis decreased starch content but increased glucose content in leaves, indicating Ibßfruct2-1 was a negative regulator of starch content. These findings represent an important advance in understanding the regulatory and functional divergence among duplicated genes in sweet potato, and provide critical information for functional studies and utilization of these genes in genetic improvement.
RESUMEN
As a key enzyme in the starch and sugar metabolic pathways in sweet potato (Ipomoea batatas (L.) Lam.), the vacuolar invertase (EC 3.2.1.26) IbßFRUCT2 is involved in partitioning and modulating the starch and sugar components of the storage root. However, the post-translational regulation of its invertase activity remains unclear. In this study, we identified three invertase inhibitors, IbInvInh1, IbInvInh2, and IbInvInh3, as potential interaction partners of IbßFRUCT2. All were found to act as vacuolar invertase inhibitors (VIFs) and belonged to the plant invertase/pectin methyl esterase inhibitor superfamily. Among the three VIFs, IbInvInh2 is a novel VIF in sweet potato and was confirmed to be an inhibitor of IbßFRUCT2. The N-terminal domain of IbßFRUCT2 and the Thr39 and Leu198 sites of IbInvInh2 were predicted to be engaged in their interactions. The transgenic expression of IbInvInh2 in Arabidopsis thaliana plants reduced the starch content of leaves, while its expression in the Ibßfruct2-expressing Arabidopsis plants increased the starch content of leaves, suggesting that the post-translational inhibition of IbßFRUCT2 activity by IbInvInh2 contributes to the regulation of the plant starch content. Taken together, our findings reveal a novel VIF in sweet potato and provide insights into the potential regulatory roles of the VIFs and invertase-VIF interaction in starch metabolism. These insights lay the foundation for using VIFs to improve the starch properties of crops.