Quality Control Of Membrane Proteins Research Articles

Structural insights into the membrane-bound proteolytic machinery of bacterial protein quality control.

In bacteria and eukaryotic organelles of prokaryotic origin, ATP-dependent proteases are crucial for regulating protein quality control through substrate unfolding and degradation. Understanding the mechanism and regulation of this key cellular process could prove instrumental in developing therapeutic strategies. Very recently, cryo-electron microscopy structural studies have shed light on the functioning of AAA+ proteases, including membrane-bound proteolytic complexes. This review summarizes the structure and function relationship of bacterial AAA+ proteases, with a special focus on the sole membrane-bound AAA+ protease in Escherichia coli, FtsH. FtsH substrates include both soluble cytoplasmic and membrane-incorporated proteins, highlighting its intricate substrate recognition and processing mechanisms. Notably, 12 copies of regulatory HflK and HflC proteins, arranged in a cage-like structure embedded in the bacterial inner membrane, can encase up to 4 FtsH hexamers, thereby regulating their role in membrane protein quality control. FtsH represents an intriguing example, highlighting both its similarity to cytosolic AAA+ proteases with respect to overall architecture and oligomerization as well as its unique features, foremost its incorporation into a membrane-bound complex formed by HflK and HflC to mediate its function in protein quality control.

The Dsc complex and its role in Golgi quality control.

Membrane proteins play crucial roles in cellular functions. However, processes such as the insertion of membrane proteins into the endoplasmic reticulum (ER), their folding into native structures, the assembly of multi-subunit membrane protein complexes, and their targeting from the ER to specific organelles are prone to errors and have a relatively high failure rate. To prevent the accumulation of defective or orphaned membrane proteins, quality control mechanisms assess folding, quantity, and localization of these proteins. This quality control is vital for preserving organelle integrity and maintaining cellular health. In this mini-review, we will focus on how selective membrane protein quality control at the Golgi apparatus, particularly through the defective for SREBP cleavage (Dsc) ubiquitin ligase complex, detects orphaned proteins and prevents their mis-localization to other organelles.

The Role of FtsH Complexes in the Response to Abiotic Stress in Cyanobacteria.

FtsH proteases (FtsHs) belong to intramembrane ATP-dependent metalloproteases which are widely distributed in eubacteria, mitochondria and chloroplasts. The best-studied roles of FtsH in Escherichia coli include quality control of membrane proteins, regulation of response to heat shock, superoxide stress and viral infection, and control of lipopolysaccharide biosynthesis. While heterotrophic bacteria mostly contain a single indispensable FtsH complex, photosynthetic cyanobacteria usually contain three FtsH complexes: two heterocomplexes and one homocomplex. The essential cytoplasmic FtsH1/3 most probably fulfills a role similar to other bacterial FtsHs, whereas the thylakoid FtsH2/3 heterocomplex and FtsH4 homocomplex appear to maintain the photosynthetic apparatus of cyanobacteria and optimize its functionality. Moreover, recent studies suggest the involvement of all FtsH proteases in a complex response to nutrient stresses. In this review, we aim to comprehensively evaluate the functions of the cyanobacterial FtsHs specifically under stress conditions with emphasis on nutrient deficiency and high irradiance. We also point to various unresolved issues concerning FtsH functions, which deserve further attention.

ER membrane complex (EMC): Structure, functions, and roles in diseases.

The endoplasmic reticulum (ER) is the largest membrane system in eukaryotic cells and is the primary site for the biosynthesis of lipids and carbohydrates, as well as for the folding, assembly, modification, and transport of secreted and integrated membrane proteins. The ER membrane complex (EMC) on the ER membrane is an ER multiprotein complex that affects the quality control of membrane proteins, which is abundant and widely preserved. Its disruption has been found to affect a wide range of processes, including protein and lipid synthesis, organelle communication, endoplasmic reticulum stress, and viral maturation, and may lead to neurodevelopmental disorders and cancer. Therefore, EMC has attracted the attention of many scholars and become a hot field. In this paper, we summarized the main contributions of the research of EMC in the past nearly 15 years, and reviewed the structure and function of EMC as well as its related diseases. We hope this review will promote further progress of research on EMC.

TOLLIP acts as a cargo adaptor to promote lysosomal degradation of aberrant ER membrane proteins.

Endoplasmic reticulum (ER) proteostasis is maintained by various catabolic pathways. Lysosomes clear entire ER portions by ER-phagy, while proteasomes selectively clear misfolded or surplus aberrant proteins by ER-associated degradation (ERAD). Recently, lysosomes have also been implicated in the selective clearance of aberrant ER proteins, but the molecular basis remains unclear. Here, we show that the phosphatidylinositol-3-phosphate (PI3P)-binding proteinTOLLIP promotes selective lysosomal degradation of aberrant membrane proteins, including an artificial substrate and motoneuron disease-causing mutants of VAPB and Seipin. These cargos are recognized by TOLLIP through its misfolding-sensing intrinsically disordered region (IDR) and ubiquitin-binding CUE domain. In contrast to ER-phagy receptors, which clear both native and aberrant proteins by ER-phagy, TOLLIP selectively clears aberrant cargos by coupling them with the PI3P-dependent lysosomal trafficking without promoting bulk ER turnover. Moreover, TOLLIP depletion augments ER stress after ERAD inhibition, indicating that TOLLIP and ERAD cooperatively safeguard ER proteostasis. Our study identifies TOLLIP as a unique type of cargo-specific adaptor dedicated to the clearance of aberrant ER cargos and provides insights into molecular mechanisms underlying lysosome-mediated quality control of membrane proteins.

P97/VCP Promotes the Recycling of Endocytic Cargo.

The endocytic pathway is of central importance for eukaryotic cells, as it enables uptake of extracellular materials, membrane protein quality control and recycling, as well as modulation of receptor signaling. While the ATPase p97 (VCP, Cdc48) has been found to be involved in the fusion of early endosomes and endolysosomal degradation, its role in endocytic trafficking is still incompletely characterized. Here, we identify myoferlin (MYOF), a ferlin family member with functions in membrane trafficking and repair, as a hitherto unknown p97 interactor. The interaction of MYOF with p97 depends on the cofactor PLAA previously linked to endosomal sorting. Besides PLAA, shared interactors of p97 and MYOF comprise several proteins involved in endosomal recycling pathways, including Rab11, Rab14, and the transferrin receptor CD71. Accordingly, a fraction of p97 and PLAA localizes to MYOF-, Rab11-, and Rab14-positive endosomal compartments. Pharmacological inhibition of p97 delays transferrin recycling, indicating that p97 promotes not only the lysosomal degradation, but also the recycling of endocytic cargo.

Chloroplast SRP54 and FtsH protease coordinate thylakoid membrane-associated proteostasis in Arabidopsis.

Thylakoid membrane protein quality control (PQC), which requires the coordination of membrane protein translocation and degradation of unassembled proteins, determines chloroplast development during de-etiolation. Despite numerous efforts, the regulation of this process in land plants is largely unknown. Here, we report the isolation and characterization of pale green Arabidopsis4 (pga4) mutants in Arabidopsis (Arabidopsis thaliana) with defects in chloroplast development during de-etiolation. Map-based cloning and complementation assays confirmed that PGA4 encodes the chloroplast Signal Recognition Particle 54 kDa (cpSRP54) protein. A heterogeneous Light-Harvesting Chlorophyll a/b Binding-Green Fluorescent Protein (LhcB2-GFP) fusion protein was generated as an indicative reporter for cpSRP54-mediated thylakoid translocation. LhcB2-GFP was dysfunctional and degraded to a short-form dLhcB2-GFP during de-etiolation through an N-terminal degradation initiated on thylakoid membranes. Further biochemical and genetic evidence demonstrated that the degradation of LhcB2-GFP to dLhcB2-GFP was disrupted in pga4 and yellow variegated2 (var2) mutants caused by mutations in the Filamentous Temperature-Sensitive H2 (VAR2/AtFtsH2) subunit of thylakoid FtsH. The yeast two-hybrid assay showed that the N-terminus of LhcB2-GFP interacts with the protease domain of VAR2/AtFtsH2. Moreover, the over-accumulated LhcB2-GFP in pga4 and var2 formed protein aggregates, which were insoluble in mild nonionic detergents. Genetically, cpSRP54 is a suppressor locus for the leaf variegation phenotype of var2. Together, these results demonstrate the coordination of cpSRP54 and thylakoid FtsH in maintaining thylakoid membrane PQC during the assembly of photosynthetic complexes and provide a trackable substrate and product for monitoring cpSRP54-dependent protein translocation and FtsH-dependent protein degradation.

The ERAD system is restricted by elevated ceramides.

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as ER-associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, and ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition influences ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very-long-chain ceramides leads to a marked increase in the level of ubiquitinated substrates in the ER membrane and concomitantly reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized mechanism in which ER membrane lipid remodeling changes the activity of ERAD.

Volleying plasma membrane proteins from birth to death: Role of J-domain proteins.

The function, stability, and turnover of plasma membrane (PM) proteins are crucial for cellular homeostasis. Compared to soluble proteins, quality control of plasma membrane proteins is extremely challenging. Failure to meet the high quality control standards is detrimental to cellular and organismal health. J-domain proteins (JDPs) are among the most diverse group of chaperones that collaborate with other chaperones and protein degradation machinery to oversee cellular protein quality control (PQC). Although fragmented, the available literature from different models, including yeast, mammals, and plants, suggests that JDPs assist PM proteins with their synthesis, folding, and trafficking to their destination as well as their degradation, either through endocytic or proteasomal degradation pathways. Moreover, some JDPs interact directly with the membrane to regulate the stability and/or functionality of proteins at the PM. The deconvoluted picture emerging is that PM proteins are relayed from one JDP to another throughout their life cycle, further underscoring the versatility of the Hsp70:JDP machinery in the cell.

The human signal peptidase complex acts as a quality control enzyme for membrane proteins.

Cells need to detect and degrade faulty membrane proteins to maintain homeostasis. In this study, we identify a previously unknown function of the human signal peptidase complex (SPC)-the enzyme that removes endoplasmic reticulum (ER) signal peptides-as a membrane protein quality control factor. We show that the SPC cleaves membrane proteins that fail to correctly fold or assemble into their native complexes at otherwise hidden cleavage sites, which our study reveals to be abundant in the human membrane proteome. This posttranslocational cleavage synergizes with ER-associated degradation to sustain membrane protein homeostasis and contributes to cellular fitness. Cryptic SPC cleavage sites thus serve as predetermined breaking points that, when exposed, help to target misfolded or surplus proteins for degradation, thereby maintaining a healthy membrane proteome.

The Arabidopsis NLP7-HB52/54-VAR2 pathway modulates energy utilization in diverse light and nitrogen conditions

In plants, nitrate is the dominant nitrogen (N)source and a critical nutrient signal regulating various physiological and developmental processes.1,2,3,4 Nitrate-responsive gene regulatory networks are widely believed to control growth, development, and life cycle in addition to N acquisition and utilization,1,2,3,4 and NIN-LIKE PROTEIN (NLP) transcriptional activators have been identified as the master regulators governing the networks.5,6,7 However, it remains to be elucidated how nitrate signaling regulates respective physiological and developmental processes. Here, we have identified a new nitrate-activated transcriptional cascade involved in chloroplast development and the maintenance of chloroplast function in Arabidopsis. This cascade consisting of NLP7 and two homeodomain-leucine zipper (HD-Zip) class I transcription factors, HOMEOBOX PROTEIN52 (HB52) and HB54,8,9 was responsible for nitrate- and light-dependent expression of VAR2 encoding the FtsH2 subunit of the chloroplast FtsH protease involved in the quality control of photodamaged thylakoid membrane proteins.10,11 Consistently, the nitrate-activated NLP7-HB52/54-VAR2 pathway underpinned photosynthetic light energy utilization, especially in high light environments. Furthermore, genetically enhancing the NLP7-HB52/54-VAR2 pathway resulted in improved light energy utilization under high light and low N conditions, a superior agronomic trait. These findings shed light on a new role of nitrate signaling and a novel mechanism for integrating information on N nutrient and light environments, providing a hint for enhancing the light energy utilization of plants in low N environments.

The ERAD system is controlled by ceramides in the endoplasmic reticulum membrane

Misfolded proteins in the endoplasmic reticulum (ER) are removed through a process known as endoplasmic reticulum associated degradation (ERAD). ERAD occurs through an integral membrane protein quality control system that recognizes substrates, retrotranslocates the substrates across the membrane, ubiquitinates and extracts the substrates from the membrane for degradation at the cytosolic proteasome. While ERAD systems are known to regulate lipid biosynthetic enzymes, the regulation of ERAD systems by the lipid composition of cellular membranes remains unexplored. Here, we report that the ER membrane composition controls ERAD function by incapacitating substrate extraction. Unbiased lipidomic profiling revealed that elevation of specific very‐long chain ceramides leads to a dramatic increase in the level of ubiquitinated substrates in the ER membrane, and concomitantly, reduces extracted substrates in the cytoplasm. This work reveals a previously unrecognized regulatory mechanism in which ER membrane lipid remodeling controls the ERAD system.

P97 dysfunction underlies a loss of quality control of damaged membrane proteins and promotes oxidative stress and sickling in sickle cell disease.

Sickling is the central pathogenic process of sickle cell disease (SCD), one of the most prevalent inherited hemolytic disorders. Having no easy access to antioxidants in the cytosol, elevated levels of reactive oxygen species (ROS) residing at the plasma membrane in sickle red blood cells (sRBCs) easily oxidize membrane proteins and thus contribute to sickling. Although the ubiquitin-proteasome system (UPS) is essential to rapidly clear ROS-damaged membrane proteins and maintain cellular homeostasis, the function and regulatory mechanism of the UPS for their clearance in sRBCs remains unidentified. Elevated levels of polyubiquitinated membrane-associated proteins in human sRBCs are reported here. High throughput and untargeted proteomic analyses of membrane proteins immunoprecipitated by ubiquitin antibodies detected elevated levels of ubiquitination of a series of proteins including cytoskeletal proteins, transporters, ROS-related proteins, and UPS machinery components in sRBCs. Polyubiquitination of membrane-associated catalase was increased in sRBCs, associated with decreased catalase activity and elevated ROS. Surprisingly, shuttling of p97 (ATP-dependent valosin-containing chaperone protein), a key component of the UPS to shuttle polyubiquitinated proteins from the membrane to cytosol for proteasomal degradation, was significantly impaired, resulting in significant accumulation of p97 along with polyubiquitinated proteins in the membrane of human sRBCs. Functionally, inhibition of p97 directly promoted accumulation of polyubiquitinated membrane-associated proteins, excessive ROS levels, and sickling in response to hypoxia. Overall, we revealed that p97 dysfunction underlies impaired UPS and contributes to oxidative stress in sRBCs.

Cryo-EM structure of transmembrane AAA+ protease FtsH in the ADP state

AAA+ proteases regulate numerous physiological and cellular processes through tightly regulated proteolytic cleavage of protein substrates driven by ATP hydrolysis. FtsH is the only known family of membrane-anchored AAA+ proteases essential for membrane protein quality control. Although a spiral staircase rotation mechanism for substrate translocation across the FtsH pore has been proposed, the detailed conformational changes among various states have not been clear due to absence of FtsH structures in these states. We report here the cryo-EM structure for Thermotoga maritima FtsH (TmFtsH) in a fully ADP-bound symmetric state. Comparisons of the ADP-state structure with its apo-state and a substrate-engaged yeast YME1 structure show conformational changes in the ATPase domains, rather than the protease domains. A reconstruction of the full-length TmFtsH provides structural insights for the dynamic transmembrane and the periplasmic domains. Our structural analyses expand the understanding of conformational switches between different nucleotide states in ATP hydrolysis by FtsH.

Exploring the molecular features of protein substrates determining the susceptibility to FtsH-mediated protein degradation

AAA+ proteases are universally conserved molecular machines that utilize ATP hydrolysis to actively unfold substrate proteins and translocate them into a compartmental protease for degradation. This combined activity is central to the removal of misfolded and intrinsically unstable proteins or the proteins that are no longer needed. The FtsH family enzymes are only membrane-integrated AAA+ proteases. In the membranes of bacteria, mitochondria and chloroplasts, FtsH and homologous enzymes play a pivotal role in membrane protein quality control and regulatory circuits degrading both membrane and water-soluble proteins.

Nanodisc-Based Proteomics Identify Caj1 as an Hsp40 with Affinity for Phosphatidic Acid Lipids.

Many soluble proteins interact with membranes to perform important biological functions, including signal transduction, regulation, transport, trafficking, and biogenesis. Despite their importance, these protein-membrane interactions are difficult to characterize due to their often-transient nature as well as phospholipids' poor solubility in aqueous solution. Here, we employ nanodiscs-small, water-soluble patches of a lipid bilayer encircled with amphipathic scaffold proteins-along with quantitative proteomics to identify lipid-binding proteins in Saccharomyces cerevisiae. Using nanodiscs reconstituted with yeast total lipid extracts or only phosphatidylethanolamine (PE-nanodiscs), we capture several known membrane-interacting proteins, including the Rab GTPases Sec4 and Ypt1, which play key roles in vesicle trafficking. Utilizing PE-nanodiscs enriched with phosphatidic acid (PEPA-nanodiscs), we specifically capture a member of the Hsp40/J-protein family, Caj1, whose function has recently been linked to membrane protein quality control. We show that the Caj1 interaction with liposomes containing PA is modulated by pH and PE lipids and depends on two patches of positively charged residues near the C-terminus of the protein. The protein Caj1 is the first example of an Hsp40/J-domain protein with affinity for membranes and phosphatidic acid lipid specificity. These findings highlight the utility of combining proteomics with lipid nanodiscs to identify and characterize protein-lipid interactions that may not be evident using other methods. Data are available via ProteomeXchange with the identifier PXD027992.

Allosteric activation of Tcell antigen receptor signaling by quaternary structure relaxation.

SummaryThe mechanism of T cell antigen receptor (TCR-CD3) signaling remains elusive. Here, we identify mutations in the transmembrane region of TCRβ or CD3ζ that augment peptide T cell antigen receptor (pMHC)-induced signaling not explicable by enhanced ligand binding, lateral diffusion, clustering, or co-receptor function. Using a biochemical assay and molecular dynamics simulation, we demonstrate that the gain-of-function mutations loosen the interaction between TCRαβ and CD3ζ. Similar to the activating mutations, pMHC binding reduces TCRαβ cohesion with CD3ζ. This event occurs prior to CD3ζ phosphorylation and at 0°C. Moreover, we demonstrate that soluble monovalent pMHC alone induces signaling and reduces TCRαβ cohesion with CD3ζ in membrane-bound or solubilised TCR-CD3. Our data provide compelling evidence that pMHC binding suffices to activate allosteric changes propagating from TCRαβ to the CD3 subunits, reconfiguring interchain transmembrane region interactions. These dynamic modifications could change the arrangement of TCR-CD3 boundary lipids to license CD3ζ phosphorylation and initiate signal propagation.

The ER membrane protein complex subunit Emc3 controls angiogenesis via the FZD4/WNT signaling axis.

The endoplasmic reticulum (ER) membrane protein complex (EMC) regulates the synthesis and quality control of membrane proteins with multiple transmembrane domains. One of the membrane spanning subunits, EMC3, is a core member of the EMC complex that provides essential hydrophilic vestibule for substrate insertion. Here, we show that the EMC subunit Emc3 plays critical roles in the retinal vascular angiogenesis by regulating Norrin/Wnt signaling. Postnatal endothelial cell (EC)-specific deletion of Emc3 led to retarded retinal vascular development with a hyperpruned vascular network, the appearance of blunt-ended, aneurysm-like tip endothelial cells (ECs) with reduced numbers of filopodia and leakage of erythrocytes at the vascular front. Diminished tube formation and cell proliferation were also observed in EMC3 depleted human retinal endothelial cells (HRECs). We then discovered a critical role for EMC3 in expression of FZD4 receptor of β-catenin signaling using RNA sequencing, real-time quantitative PCR (RT-qPCR) and luciferase reporter assay. Moreover, augmentation of Wnt activity via lithium chloride (LiCl) treatment remarkably enhanced β-catenin signaling and cell proliferation of HRECs. Additionally, LiCl partially reversed the angiogenesis defects in Emc3-cKO mice. Our data reveal that Emc3 plays essential roles in angiogenesis through direct control of FZD4 expression and Norrin/β-catenin signaling.

Membrane Protein Quality Control Mechanisms in the Endo-Lysosome System.

Protein quality control (PQC) machineries play a critical role in selective identification and removal of mistargeted, misfolded, and aberrant proteins. This task is extremely complicated due to the enormous diversity of the proteome. It also requires nuanced and careful differentiation between 'normal' and 'folding intermediates' from 'abnormal' and 'misfolded' protein states. Multiple genetic and proteomic approaches have started to delineate the molecular underpinnings of how these machineries recognize their target and how their activity is regulated. In this review, we summarize our understanding of the various E3 ubiquitin ligases and associated machinery that mediate PQC in the endo-lysosome system in yeast and humans, how they are regulated, and mechanisms of target selection, with the intent of guiding future research in this area.

The cytoplasmic domain of the AAA+ protease FtsH is tilted with respect to the membrane to facilitate substrate entry

AAA+ proteases are degradation machines that use ATP hydrolysis to unfold protein substrates and translocate them through a central pore toward a degradation chamber. FtsH, a bacterial membrane-anchored AAA+ protease, plays a vital role in membrane protein quality control. How substrates reach the FtsH central pore is an open key question that is not resolved by the available atomic structures of cytoplasmic and periplasmic domains. In this work, we used both negative stain TEM and cryo-EM to determine 3D maps of the full-length Aquifex aeolicus FtsH protease. Unexpectedly, we observed that detergent solubilization induces the formation of fully active FtsH dodecamers, which consist of two FtsH hexamers in a single detergent micelle. The striking tilted conformation of the cytosolic domain in the FtsH dodecamer visualized by negative stain TEM suggests a lateral substrate entrance between the membrane and cytosolic domain. Such a substrate path was then resolved in the cryo-EM structure of the FtsH hexamer. By mapping the available structural information and structure predictions for the transmembrane helices to the amino acid sequence we identified a linker of ∼20 residues between the second transmembrane helix and the cytosolic domain. This unique polypeptide appears to be highly flexible and turned out to be essential for proper functioning of FtsH as its deletion fully eliminated the proteolytic activity of FtsH.