Highlights d Dfm1 selectively binds ERAD-targeted membrane substrates d Polyubiquitin chains bind directly to Cdc48 recruited by Dfm1 d Derlin lipid thinning facilitates removal of integral membrane substrates in the ER d Substrate engagement and lipid thinning are conserved derlin features

Oxygenic respiration and photosynthesis based on quinone redox reactions face a danger of wasteful energy dissipation by diversion of the productive electron transfer pathway through the generation of reactive oxygen species (ROS). Nevertheless, the widespread quinone oxido-reductases from the cytochrome bc family limit the amounts of released ROS to a low, perhaps just signaling, level through an as-yet-unknown mechanism. Here, we propose that a metastable radical state, nonreactive with oxygen, safely holds electrons at a local energetic minimum during the oxidation of plastohydroquinone catalyzed by the chloroplast cytochrome b 6 f. This intermediate state is formed by interaction of a radical with a metal cofactor of a catalytic site. Modulation of its energy level on the energy landscape in photosynthetic vs. respiratory enzymes provides a possible mechanism to adjust electron transfer rates for efficient catalysis under different oxygen tensions.cytochrome b 6 f | reactive oxygen species | semiquinone | electron paramagnetic resonance | electron transport P hotosynthetic and respiratory cytochromes bc 1 /b 6 f (Fig. 1A) generate a proton-motive force (pmf) that powers cellular metabolism by using the Gibbs free energy difference (ΔG) between hydroquinone (QH 2 ) derivatives (Fig. 1B) and oxidized soluble electron transfer proteins (e.g., cytochrome c or plastocyanin) (1, 2). To increase the efficiency of this process, which is critical for the yield of the generated pmf, one part of the enzyme recirculates electrons to the quinone pool in the membrane (Q pool), whereas the second part steers the electrons to the cytochrome c pool, powering the electron recirculation (Fig. 1C). This mechanism, which is best established for the cytochrome bc 1 (cyt bc 1 ) (3, 4), with supporting data for the cytochrome b 6 f (cyt b 6 f) (5), discussed in ref. 2, is based on bifurcation of the route for two electrons released upon oxidation of QH 2 at one of the catalytic sites-the Q p site, (Q p ), (Fig. 1D) (3-5). A model for the energetics of this reaction assumes that one electron derived from the two-electron QH 2 donor is transferred, through the high-potential cofactor chain ("steering part" in Fig. 1C) to plastocyanin or cytochrome c, whereas the second electron is transferred across the membrane through low-potential cofactors ("recirculation" part in Fig. 1C).The electronic bifurcation process requires formation of a short-lived and reducing redox intermediate-ubisemiquinone (USQ) or plastosemiquinone (PSQ) (4, 6, 7). However, such an intermediate in an oxygenic environment would readily reduce oxygen to form superoxide radical, (O 2 − ), compromising the efficiency of energy conservation (8). Even in cyt b 6 f where the level of superoxide production through this pathway is at least an order of magnitude greater than that from yeast cyt bc 1 , the branching ratio for electron transfer to O 2 forming O 2 − is only 1-2% of the total flux (6). The low absolute level of O 2 − production in native proteins implies the e...

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The intramembrane cytochrome bc complex of the photosynthetic bacterium Rhodobacter capsulatus and the cytochrome bf complex, which functions in oxygenic photosynthesis, utilize two pairs of b-hemes in a symmetric dimer to accomplish proton-coupled electron transfer. The transmembrane electron transfer pathway in each complex was identified through the novel use of heme Soret band excitonic circular dichroism (CD) spectra, for which the responsible heme-heme interactions were determined from crystal structures. Kinetics of heme reduction and CD amplitude change were measured simultaneously. For bc, in which the redox potentials of the transmembrane heme pair are separated by 160 mV, heme reduction occurs preferentially to the higher-potential intermonomer heme pair on the electronegative (n) side of the complex. This contrasts with the bf complex, where the redox potential difference between transmembrane intramonomer p- and n-side hemes is substantially smaller and the n-p pair is preferentially reduced. Limits on the dielectric constant between intramonomer hemes were calculated from the interheme distance and the redox potential difference, ΔE. The difference in preferred reduction pathway is a consequence of the larger ΔE between n- and p-side hemes in bc, which favors the reduction of n-side hemes and cannot be offset by decreased repulsive Coulombic interactions between intramonomer hemes.

Several ‘super-complexes’ of individual hetero-oligomeric membrane protein complexes, whose function is to facilitate intra-membrane electron and proton transfer and harvesting of light energy, have been previously characterized in the mitochondrial cristae and chloroplast thylakoid membranes. We report the presence of an intra-membrane super-complex dominated by the ATP-synthase, photosystem I (PSI) reaction-center complex and the ferredoxin-NADP + Reductase (FNR) in the thylakoid membrane. The presence of the super-complex has been documented by mass spectrometry, clear-native PAGE and Western Blot analyses. This is the first documented presence of ATP synthase in a super-complex with the PSI reaction-center located in the non-appressed stromal domain of the thylakoid membrane.

Nearly one-third of nascent proteins are initially targeted to the endoplasmic reticulum (ER), where they are correctly folded and assembled before being delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ERassociated degradation (ERAD) removes these client proteins from the ER membrane to the cytosol in a process known as retrotranslocation. Our previous work demonstrated that rhomboid pseudoprotease Dfm1 is involved in the retrotranslocation of ubiquitinated membrane integral ERAD substrates. Herein, we found that Dfm1 associates with the SPOTS complex, which is composed of serine palmitoyltransferase (SPT) enzymes and accessory components that are critical for catalyzing the first rate-limiting step of the sphingolipid biosynthesis pathway. Furthermore, Dfm1 employs an ERADindependent role for facilitating the ER export and endosome-and Golgi-associated degradation (EGAD) of Orm2, which is a major antagonist of SPT activity. Given that the accumulation of human Orm2 homologs, ORMDLs, is associated with various pathologies, our study serves as a molecular foothold for understanding how dysregulation of sphingolipid metabolism leads to various diseases.

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