Membrane Contact Sites in Proteostasis and ER Stress Response

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.

Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209-220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.

Chapter Five - Protein Quality Control, Retention, and Degradation at the Endoplasmic Reticulum

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

Proteins destined for secretion in eukaryotic cells enter the endoplasmic reticulum (ER) in an unfolded state and are properly folded in this organelle and sent to their final destination. Misfolded or orphan proteins are retained in the ER by a quality control system, retrotranslocated into the cytosol and degraded. Soluble and membrane proteins were found to require a basic machinery for elimination. It is composed of (1) the E1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes, which polyubiquitinate the substrate proteins during retrotranslocation; (2) the trimeric AAA-ATPase complex Cdc48-Ufd1-Npl4p, which liberates the polyubiquitinated proteins from the ER; and (3) the 26S proteasome, finally degrading the misfolded proteins. Additional components for degradation of soluble or membrane proteins may vary depending on the nature of malfolded proteins. It is therefore of utmost importance to gain insight into the different components of the ER protein quality control and degradation system required for the elimination of the substrate variety. Protein quality control of the ER and subsequent degradation are evolutionarily highly conserved from yeast to human. The yeast Saccharomyces cerevisiae is therefore an elegant model organism for a search of new components of the ER quality control and degradation machinery, because it is easily amenable to genetic and molecular biological experimentation. In this chapter, a genetic approach is presented, which leads to the isolation of mutants and to the identification of proteins involved in protein quality control and ER-associated degradation (ERAD). The method resides in ethylmethane sulfonate (EMS) mutagenesis of a yeast strain followed by screening for stabilization of soluble ERAD substrates, two mutated and consequently malfolded vacuolar enzymes, carboxypeptidase yscY (CPY*) and proteinase yscA (PrA*). Both malfolded proteins are retained in the ER lumen and become substrates of the ERAD machinery.

Chapter 13 - Defense Against Proteotoxic Stress in the Heart: Role of p62, Autophagy, and Ubiquitin–Proteasome System

Have you HRD? Understanding ERAD Is DOAble!

Membrane proteins are essential for bacterial survival, facilitating vital processes such as energy production, nutrient transport, and cell wall synthesis. YidC is a key player in membrane protein biogenesis, acting as both an insertase and a chaperone to ensure proper protein folding and integration into the lipid bilayer. Its conserved structure and adaptability enable it to mediate co-translational and post-translational protein insertion into the membrane through both Sec-dependent and Sec-independent pathways. In addition to facilitating protein insertion, YidC collaborates with FtsH in protein quality control, preventing the accumulation of misfolded proteins that could impair cellular function. This important relationship between YidC and FtsH is poorly understood, and there is a need for further investigation into their collaboration. Understanding how YidC and FtsH coordinate their roles could provide valuable insights into the links between bacterial membrane protein biogenesis and quality control pathways. Moreover, given its central functions, YidC represents a potential target for antimicrobial development. Small molecules disrupting its function in protein folding and insertion, hold promise. However, achieving bacterial specificity without impacting eukaryotic homologs remains a challenge. Here, we review our current understanding of YidC's structure, molecular function in membrane protein biogenesis and quality control, known interactions and its therapeutic potential.

STT3B-Dependent Posttranslational N-Glycosylation as a Surveillance System for Secretory Protein

Efficient protein folding and quality control in the endoplasmic reticulum (ER) require that disulphide bonds are formed in nascent proteins, isomerised during assisted folding and reduced in terminally misfolded molecules. Recent findings in yeast and mammalian cells indicate that specific protein-protein interactions underlie redox control in the ER, allowing these competing reactions to occur simultaneously during protein quality control.

Yos9 Protein Is Essential for Degradation of Misfolded Glycoproteins and May Function as Lectin in ERAD

We recently reported the importance of Synoviolin in quality control of proteins through the endoplasmic reticulum (ER)-associated degradation (ERAD) system and its involvement in the pathogenesis of arthropathy through its anti-apoptotic effect. For further understanding of the role of Synoviolin in vivo, we generated in this study synoviolin-deficient (syno(-/-)) mice by genetargeted disruption. Strikingly, all fetuses lacking syno died in utero around embryonic day 13.5, although Hrd1p, a yeast orthologue of Synoviolin, is non-essential for survival. Histologically, hypocellularity and aberrant apoptosis were noted in the syno(-/-) fetal liver. Moreover, definitive erythropoiesis was affected in non-cell autonomous manner in syno(-/-) embryos, causing death in utero. Cultured embryonic fibroblasts derived from syno(-/-) mice were more susceptible to endoplasmic reticulum stress-induced apoptosis than those from syno(+/+) mice, but the susceptibility was rescued by overexpression of synoviolin. Our findings emphasized the indispensable role of the Synoviolin in embryogenesis.

A cell and its world of molecular machines and organelles is complex—and imperfect, full of small errors and looming catastrophes. It is challenged by stresses, environmental insults and general entropy, but works remarkably well. One reason for this is the existence of quality control. Quality control acts at the level of the important macromolecules, proteins, RNA and DNA, as well as at the organelle and whole‐cell level. It serves to edit mistakes and generally maintain functionality. Biological processes are allowed to be a bit messy and slightly unreliable as long as quality control exists. This focus in The EMBO Journal presents eight reviews, which relate to quality control in molecular and cellular biology. Some reviews are directly about quality control mechanisms, while others are more tangentially connected to it. Some of the processes discussed are clearly appreciated as ‘quality control’ and the term is frequently used, for example in ER quality control. Other quality control processes have other names, but they have in common that they serve to check for correctness and functionality of already produced macromolecules or organelles. For the quite diverse types of quality control, there are general questions that allow some comparison. First, what is being detected? Does the cell detect the healthy and correct molecule/complex/organelle and allow its persistence? Or is the unhealthy and incorrect version detected and removed? This dichotomy is related to whether degradation or persistence is the default behavior. Second, how is correctness versus incorrectness detected? Is it qualitatively or quantitatively different? Third, how are the mistakes dealt with? Are they corrected/remodeled or are they removed/degraded? And finally, what happens if quality control does not function well? Several of the reviews explore the relationship between quality control and human disease conditions. The purpose of bringing together reviews from experts in seemingly unrelated areas, …

Protein homeostasis networks in physiology and disease

Gene Therapy Strategies to Restore ER Proteostasis in Disease.