Zinc transporters take up/release zinc ions (Zn
) across biological membranes and maintain intracellular and intra-organellar Zn
homeostasis. Since this process requires a series of conformational ...changes in the transporters, detailed information about the structures of different reaction intermediates is required for a comprehensive understanding of their Zn
transport mechanisms. Recently, various Zn
transport systems have been identified in bacteria, yeasts, plants, and humans. Based on structural analyses of human ZnT7, human ZnT8, and bacterial YiiP, we propose updated models explaining their mechanisms of action to ensure efficient Zn
transport. We place particular focus on the mechanistic roles of the histidine-rich loop shared by several zinc transporters, which facilitates Zn
recruitment to the transmembrane Zn
-binding site. This review provides an extensive overview of the structures, mechanisms, and physiological functions of zinc transporters in different biological kingdoms.
Sarco/endoplasmic reticulum Ca2+ ATPase 2b (SERCA2b), a member of the SERCA family, is expressed ubiquitously and transports Ca2+ into the sarco/endoplasmic reticulum using the energy provided by ATP ...binding and hydrolysis. The crystal structure of SERCA2b in its Ca2+‐ and ATP‐bound (E1∙2Ca2+‐ATP) state and cryo‐electron microscopy (cryo‐EM) structures of the protein in its E1∙2Ca2+‐ATP and Ca2+‐unbound phosphorylated (E2P) states have provided essential insights into how the overall conformation and ATPase activity of SERCA2b is regulated by the transmembrane helix 11 and the subsequent luminal extension loop, both of which are specific to this isoform. More recently, our cryo‐EM analysis has revealed that SERCA2b likely adopts open and closed conformations of the cytosolic domains in the Ca2+‐bound but ATP‐free (E1∙2Ca2+) state, and that the closed conformation represents a state immediately prior to ATP binding. This review article summarizes the unique mechanisms underlying the conformational and functional regulation of SERCA2b.
Recent cryo‐electron microscopy analyses revealed mechanisms of structural and functional regulation of SERCA2b by its specific 11th transmembrane helix (TM11) and luminal extension tail (LE). During the SERCA catalytic cycle, ATP binding and hydrolysis in the cytosolic domains are closely coupled to Ca2+ binding and release in the transmembrane domain.
The endoplasmic reticulum (ER) is an essential cellular compartment in which an enormous number of secretory and cell surface membrane proteins are synthesized and subjected to cotranslational or ...posttranslational modifications, such as glycosylation and disulfide bond formation. Proper maintenance of ER protein homeostasis (sometimes termed proteostasis) is essential to avoid cellular stresses and diseases caused by abnormal proteins. Accumulating knowledge of cysteine-based redox reactions catalyzed by members of the protein disulfide isomerase (PDI) family has revealed that these enzymes play pivotal roles in productive protein folding accompanied by disulfide formation, as well as efficient ER-associated degradation accompanied by disulfide reduction. Each of PDI family members forms a protein–protein interaction with a preferential partner to fulfill a distinct function. Multiple redox pathways that utilize PDIs appear to function synergistically to attain the highest quality and productivity of the ER, even under various stress conditions. This review describes the structures, physiological functions, and cooperative actions of several essential PDIs, and provides important insights into the elaborate proteostatic mechanisms that have evolved in the extremely active and stress-sensitive ER.
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•PDI family members work distinctly but cooperatively to ensure proteostasis.•Structures of the PDI family provide mechanistic insights into the ER homeostasis.•Structure analyses of the PDI family with their partners will be the milestones.•New physiological roles of the PDI family will be a frontier.
Time-resolved single-molecule observations by high-speed atomic force microscopy (HS-AFM), have greatly advanced our understanding of how proteins operate to fulfill their unique functions. Using ...this device, we succeeded in visualizing two members of the protein disulfide isomerase family (PDIs) that act to catalyze oxidative folding and reductive unfolding in the endoplasmic reticulum (ER). ERdj5, an ER-resident disulfide reductase that promotes ER-associated degradation, reduces nonnative disulfide bonds of misfolded proteins utilizing the dynamics of its N-terminal and C-terminal clusters. With unfolded substrates, canonical PDI assembles to form a face-to-face dimer with a central hydrophobic cavity and multiple redox-active sites to accelerate oxidative folding inside the cavity. Altogether, PDIs exert highly dynamic mechanisms to ensure the protein quality control in the ER.
Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase ...(PDI)-the most versatile disulfide-introducing enzyme in the endoplasmic reticulum-during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates.
Almost all organisms, from bacteria to humans, possess catalytic systems that promote disulfide bond formation‐coupled protein folding, i.e. oxidative protein folding. These systems are necessary for ...the biosynthesis of many secretory and membrane proteins, such as antibodies, major histocompatibility complex molecules, growth factors, and insulin. Over the last decade, structural studies have made striking progress in this field of research, identifying how oxidative systems operate in a specific and regulated manner to maintain redox and protein homeostasis within cells. Interestingly, more and more novel catalysts that promote disulfide bond formation have been discovered in mammals, suggesting that the oxidative protein folding network is even more complicated in higher eukaryotes than previously thought. This review highlights the physiological roles and molecular bases of the disulfide bond formation pathways that have evolved in the bacterial periplasm and the endoplasmic reticulum of fungi and mammals. Accumulating knowledge about disulfide bond formation networks widely distributed throughout the biological kingdom has significantly advanced our understanding of the cellular mechanisms dedicated to protein quality control.
To catalyze oxidative folding of secretory and membrane proteins, several disulfide bond‐formation pathways exist in the endoplasmic reticulum. Ero1α and Prx4 are representative mammalian enzymes that oxidize PDI family member proteins. We review physiological roles and molecular bases of the disulfide bond formation network present in the biological kingdoms including bacteria, fungi and mammals.
Disulfide bond formation is an essential reaction involved in the folding and maturation of many secreted and membrane proteins. Both prokaryotic and eukaryotic cells utilize various disulfide ...oxidoreductases and redox-active cofactors to accelerate this oxidative reaction, and higher eukaryotes have diversified and refined these disulfide-introducing cascades over the course of evolution.
In the past decade, atomic resolution structures have been solved for an increasing number of disulfide oxidoreductases, thereby revealing the structural and mechanistic basis of cellular disulfide bond formation systems.
In this review, we focus on the evolution, structure, and regulatory mechanisms of endoplasmic reticulum oxidoreductin 1 (Ero1) family enzymes, the primary disulfide bond-generating catalysts in the endoplasmic reticulum (ER). Detailed comparison of Ero1 with other oxidoreductases, such as Prx4, QSOX, Erv1/2, and disulfide bond protein B (DsbB), provides important insight into how this ER-resident flavoenzyme acts in a regulated and specific manner to maintain redox and protein homeostasis in eukaryotic cells.
Currently, it is presumed that multiple pathways in addition to that mediated by Ero1 cooperate to achieve oxidative folding of many secretory and membrane proteins in mammalian cells. The important open question is how each oxidative pathway works distinctly or redundantly in response to various cellular conditions.
Zinc ions (Zn
) are imported into the early secretory pathway by Golgi-resident transporters, but their handling and functions are not fully understood. Here, we show that Zn
binds with high affinity ...to the pH-sensitive chaperone ERp44, modulating its localization and ability to retrieve clients like Ero1α and ERAP1 to the endoplasmic reticulum (ER). Silencing the Zn
transporters that uptake Zn
into the Golgi led to ERp44 dysfunction and increased secretion of Ero1α and ERAP1. High-resolution crystal structures of Zn
-bound ERp44 reveal that Zn
binds to a conserved histidine-cluster. The consequent large displacements of the regulatory C-terminal tail expose the substrate-binding surface and RDEL motif, ensuring client capture and retrieval. ERp44 also forms Zn
-bridged homodimers, which dissociate upon client binding. Histidine mutations in the Zn
-binding sites compromise ERp44 activity and localization. Our findings reveal a role of Zn
as a key regulator of protein quality control at the ER-Golgi interface.
Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to ...maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.
In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by approximately 20 members of the ...protein disulfide isomerase family (PDIs). PDIs consist of multiple thioredoxin-like domains and recognize a wide variety of proteins via highly conserved interdomain flexibility. Although PDIs have been studied intensely for almost 50 years, exactly how they maintain protein homeostasis in the ER remains unknown, and is important not only for fundamental biological understanding but also for protein misfolding- and aggregation-related pathophysiology. Herein, we review recent advances in structural biology and biophysical approaches that explore the underlying mechanism by which PDIs fulfil their distinct functions to promote productive protein folding and scavenge misfolded proteins in the ER, the primary factory for efficient production of the secretome.
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•Oxidative folding and chaperoning in the ER are assisted by ~20 PDI family members.•PDIs recognize and act on clients via conserved interdomain flexibility.•Exactly how PDIs maintain protein homeostasis remains to be uncovered.•Aberrant PDI activity leads to protein misfolding- and aggregation-related pathology.
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