An ERAD-independent role for rhomboid pseudoprotease Dfm1 in mediating sphingolipid homeostasis.

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, ER-associated 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 ERAD-independent 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.

The Role of the Rhomboid Superfamily in ER Protein Quality Control: From Mechanisms and Functions to Diseases.

The endoplasmic reticulum (ER) is an essential organelle in eukaryotic cells and is a major site for protein folding, modification, and lipid synthesis. Perturbations within the ER, such as protein misfolding and high demand for protein folding, lead to dysregulation of the ER protein quality control network and ER stress. Recently, the rhomboid superfamily has emerged as a critical player in ER protein quality control because it has diverse cellular functions, including ER-associated degradation (ERAD), endosome Golgi-associated degradation (EGAD), and ER preemptive quality control (ERpQC). This breadth of function both illustrates the importance of the rhomboid superfamily in health and diseases and emphasizes the necessity of understanding their mechanisms of action. Because dysregulation of rhomboid proteins has been implicated in various diseases, such as neurological disorders and cancers, they represent promising potential therapeutic drug targets. This review provides a comprehensive account of the various roles of rhomboid proteins in the context of ER protein quality control and discusses their significance in health and disease.

Derlin rhomboid pseudoproteases employ substrate engagement and lipid distortion to enable the retrotranslocation of ERAD membrane substrates.

Nearly one-third of proteins are initially targeted to the endoplasmic reticulum (ER) membrane, where they are correctly folded and then delivered to their final cellular destinations. To prevent the accumulation of misfolded membrane proteins, ER-associated degradation (ERAD) moves these clients from the ER membrane to the cytosol, a process known as retrotranslocation. Our recent work in Saccharomyces cerevisiae reveals a derlin rhomboid pseudoprotease, Dfm1, is involved in the retrotranslocation of ubiquitinated ERAD membrane substrates. In this study, we identify conserved residues of Dfm1 that are critical for retrotranslocation. We find several retrotranslocation-deficient Loop 1 mutants that display impaired binding to membrane substrates. Furthermore, Dfm1 possesses lipid thinning function to facilitate in the removal of ER membrane substrates, and this feature is conserved in its human homolog, Derlin-1, further implicating that derlin-mediated retrotranslocation is a well-conserved process.

Assays for studying normal versus suppressive ERAD-associated retrotranslocation pathways in yeast.

In S. cerevisiae, we identified rhomboid pseudoprotease Dfm1 as the major mediator for removing or retrotranslocating misfolded membrane substrates from the ER (endoplasmic reticulum). Long-standing challenges with rapid suppression of dfm1-null cells have limited the biochemical study of Dfm1's role in ER protein quality control. Here, we provide a protocol for the generation and handling of dfm1-null cells and procedures for studying normal vs. suppressive alternative retrotranslocation pathways. Our methods can be utilized to study other components involved in retrotranslocation. For complete information on the generation and use of this protocol, please refer to Neal et al. (2017, 2018); Neal et al. (2019); Neal et al. (2020).

A novel chloroplast super-complex consisting of the ATP synthase and photosystem I reaction center.

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.

Structural and functional contributions of lipids to the stability and activity of the photosynthetic cytochrome b6f lipoprotein complex.

The photosynthetic cytochrome b6f complex, a homodimer containing eight distinct subunits and 26 transmembrane helices per monomer, catalyzes proton-coupled electron transfer across the thylakoid membrane. The 2.5-Å-resolution structure of the complex from the cyanobacterium Nostoc sp. revealed the presence of 23 lipid-binding sites per monomer. Although the crystal structure of the cytochrome b6f from a plant source has not yet been solved, the identities of the lipids present in a plant b6f complex have previously been determined, indicating that the predominant lipid species are monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG), and sulfoquinovosyldiacylglycerol (SQDG). Despite the extensive structural analyses of b6f-lipid interactions, the basis of the stabilization by lipids remains poorly understood. In the present study, we report on the effect of individual lipids on the structural and functional integrity of the b6f complex, purified from Spinacea oleracea It was found that (i) galactolipids (MGDG, DGDG, and SQDG) and phospholipids dilinolenoyl-phosphatidylglycerol (DLPG), 1,2-dioleoylphosphatidylglycerol (DOPG), and 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine (DOPC) structurally stabilize the complex to varying degrees; (ii) SQDG has a major role in stabilizing the dimeric complex; (iii) the b6f complex is stabilized by incorporation into nanodiscs or bicelles; (iv) removal of bound phospholipid by phospholipase A2 inactivates the cytochrome complex; and (v) activity can be restored significantly by the addition of the anionic lipid PG, which is attributed to stabilization of the quinone portal and the hinge region of the iron-sulfur protein.

Metastable radical state, nonreactive with oxygen, is inherent to catalysis by respiratory and photosynthetic cytochromes bc1/b6f.

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 b6f 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.

Synchrotron small-angle X-ray scattering studies of hemoglobin nonaggregation confined inside polymer capsules.

The effect of confinement on the structure of hemoglobin (Hb) within polymer capsules was investigated here. Hemoglobin transformed from an aggregated state in solution to a nonaggregated state when confined inside the polymer capsules. This was directly confirmed using synchrotron small-angle X-ray scattering (SAXS) studies. The radius of gyration (R(g)) and polydispersity (p) of the proteins in the confined state were smaller compared to those in solution. In fact, the R(g) value is very similar to theoretical values obtained using protein structures generated from the Protein Databank. In the temperature range (25-85 °C, Tm 59 °C), the R(g) values for the confined Hb remained constant. This observation is in contrary to the increasing R(g) values obtained for the bare Hb in solution. This suggested higher thermal stability of Hb when confined inside the polymer capsule than when in solution. Changes in protein configuration were also reflected in the protein function. Confinement resulted in a beneficial enhancement of the electroactivity of Hb. While Hb in solution showed dominance of the cathodic process (Fe(3+) → Fe(2+)), efficient reversible Fe(3+)/Fe(2+) redox response is observed in the case of the confined Hb. This has important protein functional implications. Confinement allows the electroactive heme to take up positions favorable for various biochemical activities such as sensing of analytes of various sizes from small to macromolecules and controlled delivery of drugs.