Pro-apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site-specific disulfide crosslinking, compartment-specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3-in-groove dimer interface on the MOM surface similar to that observed in crystals. However, after the a5 helix was released into the MOM, the remaining interface with a2, a3, and a4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel a9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3-in-groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, a9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by a9 insertion into the MOM and localization to the pore rim. Therefore, the BH3-in-groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by a9 dimerization at the rim.

In , FtsLB plays a central role in the initiation of cell division, possibly transducing a signal that will eventually lead to the activation of peptidoglycan remodeling at the forming septum. The molecular mechanisms by which FtsLB operates in the divisome, however, are not understood. Here, we present a structural analysis of the FtsLB complex, performed with biophysical, computational, and methods, that establishes the organization of the transmembrane region and proximal coiled coil of the complex. FRET analysis is consistent with formation of a tetramer composed of two FtsL and two FtsB subunits. We predicted subunit contacts through co-evolutionary analysis and used them to compute a structural model of the complex. The transmembrane region of FtsLB is stabilized by hydrophobic packing and by a complex network of hydrogen bonds. The coiled coil domain probably terminates near the critical constriction control domain, which might correspond to a structural transition. The presence of strongly polar amino acids within the core of the tetrameric coiled coil suggests that the coil may split into two independent FtsQ-binding domains. The helix of FtsB is interrupted between the transmembrane and coiled coil regions by a flexible Gly-rich linker. Conversely, the data suggest that FtsL forms an uninterrupted helix across the two regions and that the integrity of this helix is indispensable for the function of the complex. The FtsL helix is thus a candidate for acting as a potential mechanical connection to communicate conformational changes between periplasmic, membrane, and cytoplasmic regions.

SUMMARY Proper maintenance of mitochondrial activity is essential for metabolic homeostasis. Widespread phosphorylation of mitochondrial proteins may be an important element of this process; yet little is known about which enzymes control mitochondrial phosphorylation, or which phosphosites have functional impact. We investigate these issues by disrupting Ptc7p—a conserved but largely uncharacterized mitochondrial matrix PP2C-type phosphatase. Loss of Ptc7p causes respiratory growth defects concomitant with elevated phosphorylation of select matrix proteins. Among these, Δptc7 yeast exhibit an increase in phosphorylation of Cit1p—the canonical citrate synthase of the tricarboxylic acid cycle—that diminishes its activity. We find that phosphorylation of S462 can eliminate Cit1p enzymatic activity likely by disrupting its proper dimerization, and that Ptc7p-driven dephosphorylation rescues Cit1p activity. Collectively, our work connects Ptc7p to an essential TCA cycle function and to additional phosphorylation events that may affect mitochondrial activity inadvertently or in a regulatory manner.

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Background Simple non-isoprenoid hydrocarbons accumulate in discrete regions of the biosphere, including within bacteria and algae as a carbon and/or energy store, and the cuticles of plants and insects, where they may protect against environmental stresses. The extracellular cuticular surfaces of the stigmatic silks of maize are rich in linear hydrocarbons and therefore provide a convenient system to study the biological origins and functions of these unique metabolites. Results To test the hypotheses that genetics and environment influence the accumulation of surface hydrocarbons on silks and to examine the breadth of metabolome compositions across diverse germplasm, cuticular hydrocarbons were analyzed on husk-encased silks and silks that emerged from the husk leaves from 32 genetically diverse maize inbred lines, most of which are commonly utilized in genetics experiments. Total hydrocarbon accumulation varied ~ 10-fold among inbred lines, and up to 5-fold between emerged and husk-encased silks. Alkenes accounted for 5-60% of the total hydrocarbon metabolome, and the majority of alkenes were monoenes with a double bond at either the 7th or 9th carbon atom of the alkyl chain. Total hydrocarbon accumulation was impacted to similar degrees by genotype and husk encasement status, whereas genotype predominantly impacted alkene composition. Only minor differences in the metabolome were observed on silks that were emerged into the external environment for 3- versus 6-days. The environmental influence on the metabolome was further investigated by growing inbred lines in 2 years, one of which was warmer and wetter. Inbred lines grown in the drier year accumulated up to 2-fold more hydrocarbons and up to a 22% higher relative abundance of alkenes. In summary, the surface hydrocarbon metabolome of silks is primarily governed by genotype and husk encasement status, with smaller impacts of environment and genotype-by-environment interactions. Conclusions This study reveals that the composition of the cuticular hydrocarbon metabolome on silks is affected significantly by genetic factors, and is therefore amenable to dissection using quantitative genetic approaches. Such studies will clarify the genetic mechanisms responsible for the accumulation of these metabolites, enabling detailed functional investigations of the diverse and complex protective roles of silk surface lipids against environmental stresses.

Colocalization single-molecule methods can provide a wealth of information concerning the ordering and dynamics of biomolecule assembly. These have been used extensively to study the pathways of spliceosome assembly in vitro. Key to these experiments is the measurement of binding times-either the dwell times of a multi-molecular interaction or times in between binding events. By analyzing hundreds of these times, many new insights into the kinetic pathways governing spliceosome assembly have been obtained. Collections of binding times are often plotted as histograms and can be fit to kinetic models using a variety of methods. Here, we describe the use of maximum likelihood methods to fit dwell time distributions without binning. In addition, we discuss several aspects of analyzing these distributions with histograms and pitfalls that can be encountered if improperly binned histograms are used. We have automated several aspects of maximum likelihood fitting of dwell time distributions in the AGATHA software package.

In Escherichia coli, an important step in the divisome assembly pathway is the recruitment of the essential cell wall synthase complex FtsWI to the division site through interactions with the regulatory FtsQLB complex. Here, we investigate a key aspect of this recruitment by characterizing the structural organization of the FtsL-FtsW interaction. Mutations in the cytoplasmic and transmembrane regions of the two proteins result in cell division defects and loss of FtsW localization to division sites. We use these in vivo results to help validate the predicted interfaces from an AlphaFold2 model for the entire FtsQLBWI complex. Given the consistency between the predicted FtsQLBWI model and our current understanding of the structure and function of the complex, we further remodeled it, seeking insight into the potential structural transitions that may lead to activation of the FtsWI complex and PG synthesis. The model suggests that FtsLB serves as a support for FtsI, placing its periplasmic domain in an extended and possibly active conformation but it is also compatible with a proposed compact and possibly inactive conformation. Additionally, we reconfigure the model into an Fts[QLBWI]2 diprotomeric state, which suggests that FtsLB may act as a central hub during assembly of the PG synthesis machinery. Finally, we propose a possible role for FtsQ in activation of this machinery, potentially by acting as a gatekeeper for the interaction between the FtsL AWI region and FtsI. We propose that this gatekeeping function depends on a hinge next to the FtsLB CCD region, which has implications for the mechanisms behind the FtsLB off/on transition that is central to cell division regulation.

The FtsLB complex is a critical regulator of bacterial cell division, acting as a switch that modulates cell wall reconstruction. Evidence indicates that FtsLB exists in either an off or on state which supports the corresponding activation state of the peptidoglycan synthase complex FtsWI. In Escherichia coli, residues within FtsLB that are critical for this activation are located in a region near the C-terminal end of the periplasmic coiled coil, raising questions about the precise role of this conserved domain in the mechanism. Here, we investigate an unusual cluster of polar amino acids occurring within the core of the coiled coil. These amino acids likely reduce the structural stability of the domain and thus may be important for governing conformational changes. We found that mutating these positions to hydrophobic residues increased the thermal stability of FtsLB but caused cell division defects, suggesting that the coiled-coil domain is an intentionally "detuned" structural element. In addition, suppressor mutations were identified within the polar cluster, indicating that the precise identity of the polar amino acids is important for fine-tuning the structural balance between the off and on states. Based on energetic and sequence propensity considerations, we propose a revised structural model of the tetrameric FtsLB (named the "Y-model") in which the periplasmic domain splits into a pair of coiled-coil branches. In this configuration, the polar amino acids participate in packing within the core, but their hydrophilic terminal moieties remain more favorably exposed to water than in the original four-helix bundle model ("I-model"). The Y-model remains well structured during molecular dynamics simulations, unlike the I-model, and satisfies all known experimental constraints. For this reason, we propose the Y-model as the configuration of the coiled coil of FtsLB and that a shift in this architecture, dependent on its marginal stability, is involved in activating the complex during the process that triggers septal cell wall reconstruction.