The free energy of water-to-interface amino acid partitioning is a major contributing factor in membrane protein folding and stability. The interface residues at the C terminus of transmembrane β-barrels form the β-signal motif required for assisted β-barrel assembly in vivo but are believed to be less important for β-barrel assembly in vitro. Here, we experimentally measured the thermodynamic contribution of all 20 amino acids at the β-signal motif to the unassisted folding of the model β-barrel protein PagP. We obtained the partitioning free energy for all 20 amino acids at the lipid-facing interface (ΔΔG0w,i(φ)) and the protein-facing interface (ΔΔG0w,i(π)) residues and found that hydrophobic amino acids are most favorably transferred to the lipid-facing interface, whereas charged and polar groups display the highest partitioning energy. Furthermore, the change in non-polar surface area correlated directly with the partitioning free energy for the lipid-facing residue and inversely with the protein-facing residue. We also demonstrate that the interface residues of the β-signal motif are vital for in vitro barrel assembly, because they exhibit a side chain–specific energetic contribution determined by the change in nonpolar accessible surface. We further establish that folding cooperativity and hydrophobic collapse are balanced at the membrane interface for optimal stability of the PagP β-barrel scaffold. We conclude that the PagP C-terminal β-signal motif influences the folding cooperativity and stability of the folded β-barrel and that the thermodynamic contributions of the lipid- and protein-facing residues in the transmembrane protein β-signal motif depend on the nature of the amino acid side chain.

Legumes play an important role in the soil nitrogen availability via symbiotic nitrogen fixation (SNF). Phosphate (Pi) deficiency severely impacts SNF because of the high Pi requirement of symbiosis. Whereas PHT1 transporters are involved in Pi uptake into nodules, it is unknown how Pi is transferred from the plant infected cells to nitrogen-fixing bacteroids. We hypothesized that Medicago truncatula genes homologous to Arabidopsis PHO1, encoding a vascular apoplastic Pi exporter, are involved in Pi transfer to bacteroids. Among the seven MtPHO1 genes present in M. truncatula, we found that two genes, namely MtPHO1.1 and MtPHO1.2, were broadly expressed across the various nodule zones in addition to the root vascular system. Expressions of MtPHO1.1 and MtPHO1.2 in Nicotiana benthamiana mediated specific Pi export. Plants with nodule-specific downregulation of both MtPHO1.1 and MtPHO1.2 were generated by RNA interference (RNAi) to examine their roles in nodule Pi homeostasis. Nodules of RNAi plants had lower Pi content and a three-fold reduction in SNF, resulting in reduced shoot growth. Whereas the rate of 33Pi uptake into nodules of RNAi plants was similar to control, transfer of 33Pi from nodule cells into bacteroids was reduced and bacteroids activated their Pi-deficiency response. Our results implicate plant MtPHO1 genes in bacteroid Pi homeostasis and SNF via the transfer of Pi from nodule infected cells to bacteroids.

No abstract

The ability of histidine to participate in a wide range of stabilizing polar interactions preferentially populates this residue in functionally important sites of proteins. Histidine possesses an amphiphilic and electrostatic nature that is essential for amino acids residing at membrane interfaces. However, the frequency of occurrence of histidine at membrane interfaces, particularly transmembrane β-barrels, is lower than those of other aromatic residues. Here, we carry out comprehensive energetic measurements using equilibrium folding of the outer membrane enzyme PagP to address the contribution of a C-terminal interface histidine to barrel stability. We show that placing histidine at the C-terminus universally destabilizes PagP by 4.0–8.0 kcal mol–1 irrespective of the neighboring residue. Spectroscopic and electrophoretic measurements indicate that the altered stability may arise from a loss of barrel compaction. Isoleucine, methionine, and valine salvage this destabilization marginally (in addition to tyrosine, which shows an exceptionally high folding free energy value), when placed at the penultimate position, at the expense of an altered folding pathway. Double-mutant cycle analysis indicates that the coupling energy between the terminal and penultimate residues in PagP-X160H161 increases when the level of intrinsic destabilization by the terminal H161 is high. Our observations that neighboring residues cannot salvage the energetic destabilization of histidine may explain why histidine is less abundant at membrane interfaces.

Polycomb Repressive Complexes (PRC1 and PRC2) regulate developmental transitions in plants. AtBMI1, a PRC1 member, represses micro RNA156 (miR156) to trigger the onset of adult phase in Arabidopsis (Arabidopsis thaliana). miR156 overexpression (OE) reduces below-ground tuber yield, but stimulates aerial tubers in potato (Solanum tuberosum ssp andigena) under short-day (SD) photoperiodic conditions. Whether PRC members could govern tuber development through photoperiod-mediated regulation of miR156 is unknown. Here, we investigated the role of two PRC proteins, StMSI1 (PRC2 member) and StBMI1-1, in potato development. In wild-type andigena plants, StMSI1 and miR156 levels increased in stolon, whereas StBMI1-1 decreased under SD conditions. StMSI1-OE and StBMI1-1-antisense (AS) lines produced pleiotropic effects, including altered leaf architecture/compounding and reduced below-ground tuber yield. Notably, these lines showed enhanced miR156 accumulation accompanied by aerial stolons and tubers from axillary nodes, similar to miR156-OE lines. Further, grafting of StMSI1-OE or StBMI1-1-AS on wild-type stock resulted in reduced root biomass and showed increased accumulation of miR156a/b and-c precursors in the roots of wild-type stocks. RNA-sequencing of axillary nodes from StMSI1-OE and StBMI1-1-AS lines revealed downregulation of auxin and brassinosteroid genes, and upregulation of cytokinin transport/signaling genes, from 1,023 differentially expressed genes shared between the two lines. Moreover, we observed downregulation of genes encoding H2A-ubiquitin ligase and StBMI1-1/3, and upregulation of Trithorax group H3K4-methyl-transferases in StMSI1-OE. Chromatin immunoprecipitation-quantitative PCR confirmed H3K27me3-mediated suppression of StBMI1-1/3, and H3K4me3-mediated activation of miR156 in StMSI1-OE plants. In summary, we show that cross talk between histone modifiers regulates miR156 and alters hormonal response during aerial tuber formation in potato under SD conditions.