Cryo-EM structures of engineered active bc1-cbb3 type CIII2CIV super-complexes and electronic communication between the complexes

Respiratory electron transport complexes are organized as individual entities or combined as large supercomplexes (SC). Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the structure of an engineered bc1-cbb3 type SC (CIII2CIV, 5.2 Å resolution) and three conformers of native CIII2 (3.3 Å resolution). The SC is active in vivo and in vitro, contains all catalytic subunits and cofactors, and two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and it conveys electrons to CIV differently than cyt c2. The successful production of a native-like functional SC and determination of its structure illustrate the characteristics of membrane-confined and membrane-external respiratory electron transport pathways in Gram-negative bacteria.

Cryo-EM Structures of Respiratory bc1-cbb3 type CIII2CIV Supercomplex and Electronic Communication Between the Complexes

Respiratory electron transport complexes are organized as individual entities or combined as large super-complexes (SC). The Gram-negative bacteria deploy a mitochondrial-like cytochrome (cyt) bc1 (Complex III, CIII2), and may have specific cbb3-type cyt c oxidases (Complex IV, CIV) instead of the canonical aa3-type CIV. Electron transfer between these complexes is mediated by soluble (c2) and membrane-anchored (cy) cyts. Here, we report the first structure of a bc1-cbb3 type SC (CIII2CIV, 5.2Å resolution) and three conformers of native CIII2 (3.3Å resolution) of functional relevance. The SC contains all catalytic subunits and cofactors as well as two extra transmembrane helices attributed to cyt cy and the assembly factor CcoH. The cyt cy is integral to SC, its cyt domain is mobile and conveys electrons to CIV differently than cyt c2. For the first time, this work establishes the structural characteristics of membrane-confined and membrane-external electron transport pathways of SCs in Gram-negative bacteria.

The Sec1/Munc18 (SM) protein Vps45 is involved in iron uptake, mitochondrial function and virulence in the pathogenic fungusCryptococcus neoformans.

ABSTRACTThe battle for iron between invading microorganisms and mammalian hosts is a pivotal determinant of the outcome of infection. The pathogenic fungus,Cryptococcus neoformans, employs multiple mechanisms to compete for iron during cryptococcosis, a disease primarily of immunocompromised hosts. In this study, we examined the role of endocytic trafficking in iron uptake by characterizing a mutant defective in the Sec1/Munc18 (SM) protein Vps45. This protein is known to regulate the machinery for vesicle trafficking and fusion via interactions with SNARE proteins. As expected, avps45deletion mutant was impaired in endocytosis and showed sensitivity to trafficking inhibitors. The mutant also showed poor growth on iron-limited media and a defect in transporting the Cfo1 ferroxidase of the high-affinity iron uptake system from the plasma membrane to the vacuole. Remarkably, we made the novel observation that Vps45 also contributes to mitochondrial function in that a Vps45-Gfp fusion protein associated with mitotracker, and avps45mutant showed enhanced sensitivity to inhibitors of electron transport complexes as well as changes in mitochondrial membrane potential. Consistent with mitochondrial function, thevps45mutant was impaired in calcium homeostasis. To assess the relevance of these defects for virulence, we examined cell surface properties of thevps45mutant and found increased sensitivity to agents that challenge cell wall integrity and antifungal drugs. A change in cell wall properties was consistent with our observation of altered capsule polysaccharide attachment, and with attenuated virulence in a mouse model of cryptococcosis. Overall, our studies reveal a novel role for Vps45-mediated trafficking for iron uptake, mitochondrial function and virulence.

Coordinated downregulation of the photosynthetic apparatus as a protective mechanism against UV exposure in the diatom Corethron hystrix

The effect of ultraviolet radiation (UVR) on photosynthetic efficiency and the resulting mechanisms against UV exposure employed by phytoplankton are not completely understood. To address this knowledge gap, we developed a novel close-coupled, wavelength-configurable platform designed to produce precise and repeatable in vitro irradiation of Corethron hystrix, a member of a genera found abundantly in the Southern Ocean where UV exposure is high. We aimed to determine its metabolic, protective, mutative, and repair mechanisms as a function of varying levels of specific electromagnetic energy. Our results show that the physiological responses to each energy level of UV have a negative linear decrease in the photosynthetic efficiency of photosystem II proportional to UV intensity, corresponding to a large increase in the turnover time of quinone re-oxidation. Gene expression changes of photosystem II related reaction center proteins D1, CP43 and CP47 showed coordinated downregulation whereas the central metabolic pathway demonstrated mixed expression of up and downregulated transcripts after UVR exposure. These results suggest that while UVR may damage photosynthetic machinery, oxidative damage may limit production of new photosynthetic and electron transport complexes as a result of UVR exposure.

In rats gestational iron deficiency does not change body fat or hepatic mitochondria in the aged offspring

Mitochondrial dysfunction and resulting changes in adiposity have been observed in the offspring of animals fed a high fat (HF) diet. As iron is an important component of the mitochondria, we have studied the offspring of female rats fed complete (Con) or iron-deficient (FeD) rations for the duration of gestation to test for similar effects. The FeD offspring were ~12% smaller at weaning and remained so because of a persistent reduction in lean tissue mass. The offspring were fed a complete (stock) diet until 52 weeks of age after which some animals from each litter were fed a HF diet for a further 12 weeks. The HF diet increased body fat when compared with animals fed the stock diet, however, prenatal iron deficiency did not change the ratio of fat:lean in either the stock or HF diet groups. The HF diet caused triglyceride to accumulate in the liver, however, there was no effect of prenatal iron deficiency. The activity of the mitochondrial electron transport complexes was similar in all groups including those challenged with a HF diet. HF feeding increased the number of copies of mitochondrial DNA and the prevalence of the D-loop mutation, however, neither parameter was affected by prenatal iron deficiency. This study shows that the effects of prenatal iron deficiency differ from other models in that there is no persistent effect on hepatic mitochondria in aged animals exposed to an increased metabolic load.

Abstract 244: Distinct Regulation of Fat and Dachsous Cadherins in Response to Arterial Injury

Molecular mechanisms that control the activities of vascular smooth muscle cells (VSMCs) in the diseased or injured arterial wall remain incompletely understood. The atypical cadherin FAT1 is prominently expressed in VSMCs after vascular injury. In recent work, we found that processing of FAT1, a type I transmembrane protein, releases its intracellular domain, the FAT1ICD; in turn, FAT1ICD fragments accumulate in mitochondria and interact with electron transport complexes I and II to restrict VSMC respiration and control cell growth and neointimal formation. We hypothesized that FAT1 processing (and therefore these VSMC activities) is controlled by extracellular ligands that may interact physically via FAT1’s extended cadherin repeat or transmembrane domains. FAT4 and Dachsous 1 (DCHS1) are leading candidates for such interactions, but these proteins have not been studied in VSMCs or vascular injury response. Accordingly, we analyzed expression profiles of FAT4 and Dachsous 1 (DCHS1) during the vascular response to injury, using a rat carotid artery balloon injury model. Interestingly, FAT4 transcripts increased transiently after injury, peaking at day 7 (6.99±0.21-fold over baseline, P<.001), in a pattern reminiscent of FAT1. In contrast, DCHS1 levels decreased by day 3 after injury (0.49±0.02-fold of baseline, P<.001), remained low through day 14, and recovered by day 30. Physical interactions of FAT4 either with DCHS1 or with FAT1 have both been reported previously; therefore, decreased DCHS1 coincident with increased FAT4 suggest an increase in FAT4 availability and/or interaction with FAT1 during injury response. How this increase in FAT4 affects FAT1 function and VSMC metabolism and growth is the subject of ongoing investigation. FAT1 induction and processing after vascular injury represent an important novel molecular mechanism by which VSMC metabolism and growth are controlled after vascular injury. Increased availability of FAT4 cadherin due to FAT4 induction together with DCHS1 downregulation after vascular injury provides a likely upstream regulatory mechanism to govern FAT1 activities.