Membrane transport proteins undergo multistep conformational changes to fulfill the transport of substrates across biological membranes. Substrate release and uptake are the most important events of these multistep reactions that accompany significant conformational changes. Thus, their relevant structural intermediates should be identified to better understand the molecular mechanism. However, their identifications have not been achieved for most transporters due to the difficulty of detecting the intermediates. Herein, we report the success of these identifications for a light-driven chloride transporter halorhodopsin (HR). We compared the time course of two flash-induced signals during a single transport cycle. One is a potential change of Cl--selective membrane, which enabled us to detect tiny Cl--concentration changes due to the Cl- release and the subsequent Cl--uptake reactions by HR. The other is the absorbance change of HR reflecting the sequential formations and decays of structural intermediates. Their comparison revealed not only the intermediates associated with the key reactions but also the presences of two additional Cl--binding sites on the Cl--transport pathways. The subsequent mutation studies identified one of the sites locating the protein surface on the releasing side. Thus, this determination also clarified the Cl--transport pathway from the initial binding site until the release to the medium.

In the past decade, evidence for numerous roles of copper (Cu) in mammalian physiology has grown exponentially. The discoveries of Cu involvement in cell signaling, autophagy, cell motility, differentiation, and regulated cell death (cuproptosis) have markedly extended the list of already known functions of Cu, such as a cofactor of essential metabolic enzymes, a protein structural component, and a regulator of protein trafficking. Novel and unexpected functions of Cu transporting proteins and enzymes have been identified, and new disorders of Cu homeostasis have been described. Significant progress has been made in the mechanistic studies of two classic disorders of Cu metabolism, Menkes disease and Wilson disease, which paved ways to novel approaches to their treatment. Discovery of cuproptosis and the role of Cu in cells metastatic growth have markedly increased interest in targeting Cu homeostatic pathways to treat cancer. In this review, we summarize the established concepts in the field of mammalian Cu physiology, and discuss how new discoveries of the past decade expand and modify these concepts. The roles of Cu in brain metabolism, in cells\' functional speciation and a recently discovered regulated cell death have attracted significant attention and are highlighted in this review.

Triglyceride-rich lipoproteins (TRLs) play essential roles in human health and disease by transporting bulk lipids into the circulation. This review summarizes the fundamental mechanisms and diverse factors governing lipoprotein production, secretion, and regulation. Emphasizing the broader implications for human health, we outline the intricate landscape of lipoprotein research and highlight the potential coordination between the biogenesis and transport of TRLs in physiology, particularly the unexpected coupling of metabolic enzymes and transport machineries. Challenges and opportunities in lipoprotein biology with respect to inherited diseases and viral infections are also discussed. Further characterization of the biogenesis and transport of TRLs will advance both basic research in lipid biology and translational medicine for metabolic diseases.

The pharmacology of amino acid transporters in the SLC6 family is poorly developed compared to that of the neurotransmitter transporters. To identify new inhibitors of the proline transporter SIT1 (SLC6A20), its expression in Xenopus laevis oocytes was optimized. Trafficking of SIT1 was augmented by co-expression of angiotensin-converting enzyme 2 (ACE2) in oocytes but there was no strict requirement for co-expression of ACE2. A pharmacophore-guided screen identified tiagabine as a potent non-competitive inhibitor of SIT1. To understand its binding mode, we determined the cryo-electron microscopy (cryo-EM) structure of ACE2-SIT1 bound with tiagabine. The inhibitor binds close to the orthosteric proline binding site, but due to its size extends into the cytosolic vestibule. This causes the transporter to adopt an inward-open conformation, in which the intracellular gate is blocked. This study provides the first structural insight into inhibition of SIT1 and generates tools for a better understanding of the ACE2-SIT1 complex. These findings may have significance for SARS-CoV-2 binding to its receptor ACE2 in human lung alveolar cells where SIT1 and ACE2 are functionally expressed.

Iron is a crucial secondary metabolite for bacterial proliferation, but its bioavailability under infection conditions is limited by the low solubility of ferric ion and the host\'s ability to sequester iron by protein chelation. In these iron limiting conditions, bacteria produce and secrete low molecular weight ferric ion chelators, siderophores, to scavenge host iron. Iron bound siderophores are recognized by surface displayed receptors and internalized by active transport preceding the liberation of the iron payload by reduction or cleavage of the siderophore. The traditional paradigms surrounding the interactions between siderophores and their corresponding receptors have relied on canonical protein-ligand binding models that do not accurately reflect the conditions experienced by siderophore binding proteins (SBPs). Research by the Raymond group suggested that a ligand displacement model does not fully describe the role of SBPs in siderophore transport where the ferric ion can be shuttled between siderophore molecules during the transport process. This work inspired further research by the Wencewicz group, which demonstrated that the Staphylococcus aureus SBP FhuD2 can catalyze the transfer of iron from the biological iron source holo-transferrin to a SBP bound iron-free siderophore. The discovery of this ferrichelatase activity represents a novel mechanism of receptor mediated active transport which raises the question: is ferrichelatase activity a unique feature of FhuD2 or a previously unappreciated hallmark of SBPs? This chapter highlights a series of protocols for the general functional characterization of SBPs and methodologies to assay ferrichelatase activity with the hopes of providing the tools to answer this question.

Herpes simplex virus 1 (HSV-1) is an alpha herpesvirus that infects a majority of the world population. The mechanisms and cellular host factors involved in the intracellular transport and exocytosis of HSV-1 particles are not fully understood. To elucidate these late steps in the replication cycle, we developed a live-cell fluorescence microscopy assay of HSV-1 virion intracellular trafficking and exocytosis. This method allows us to track individual virus particles and identify the precise moment and location of particle exocytosis using a pH-sensitive reporter. We show that HSV-1 uses the host cell\'s post-Golgi secretory pathway during egress. The small GTPase, Rab6, binds to nascent secretory vesicles at the trans-Golgi network and plays important, but non-essential, roles in vesicle traffic and exocytosis at the plasma membrane, therefore making it a useful marker of the Golgi and post-Golgi secretory pathway. We show that HSV-1 particles colocalize with Rab6a in the region of the Golgi, cotraffic with Rab6a to the cell periphery, and undergo exocytosis from Rab6a vesicles. Consistent with previous reports, we find that HSV-1 particles accumulate at preferential egress sites in infected cells. The secretory pathway mediates this preferential/polarized egress, since Rab6a vesicles accumulate near the plasma membrane similarly in uninfected cells. These data suggest that, following particle envelopment, HSV-1 egress follows a pre-existing cellular secretory pathway to exit infected cells rather than novel, virus-induced mechanisms.
OBJECTIVE: Herpes simplex virus 1 (HSV-1) infects a majority of people. It establishes a life-long latent infection and occasionally reactivates, typically causing characteristic oral or genital lesions. Rarely in healthy natural hosts, but more commonly in zoonotic infections and in elderly, newborn, or immunocompromised patients, HSV-1 can cause severe herpes encephalitis. The precise cellular mechanisms used by HSV-1 remain an important area of research. In particular, the egress pathways that newly assembled virus particles use to exit from infected cells are unclear. In this study, we used fluorescence microscopy to visualize individual virus particles exiting from cells and found that HSV-1 particles use the pre-existing cellular secretory pathway.

Nucleic Acid Nanocapsules (NANs) are nucleic acid nanostructures that radially display oligonucleotides on the surface of cross-linked surfactant micelles. Their chemical makeup affords the stimuli-responsive release of therapeutically active DNA-surfactant conjugates into the cells. While NANs have so far demonstrated the effective cytosolic delivery of their nucleic acid cargo, as seen indirectly by their gene regulation capabilities, there remain gaps in the molecular understanding of how this process happens. Herein, we examine the enzymatic degradation of NANs and confirm the identity of the DNA-surfactant conjugates formed by using mass spectrometry (MS). With surface enhanced (resonance) Raman spectroscopy (SE(R)RS), we also provide evidence that the energy-independent translocation of such DNA-surfactant conjugates is contingent upon their release from the NAN structure, which, when intact, otherwise buries the hydrophobic surfactant tail in its interior. Such information suggests a critical role of the surfactant in the lipid disruption capability of the DNA surfactant conjugates generated from degradation of the NANs. Using NANs made with different tail lengths (C12 and C10), we show that this mechanism likely holds true despite significant differences in the physical properties (i.e., critical micelle concentration (CMC), surfactants per micelle, Nagg) of the resultant particles (C12 and C10 NANs). While the total cellular uptake efficiencies of C12 and C10 NANs are similar, there were differences observed in their cellular distribution and localized trafficking, even after ensuring that the total concentration of DNA was the same for both particles. Ultimately, C10 NANs appeared less diffuse within cells and colocalized less with lysosomes over time, achieving more significant knockdown of the target gene investigated, suggesting more effective endosomal escape. These differences indicate that the surfactant assembly and disassembly properties, including the number of surfactants per particle and the CMC can have important implications for the cellular delivery efficacy of DNA micelles and surfactant conjugates.

Peroxiporins are a specialized subset of aquaporins, which are integral membrane proteins primarily known for facilitating water transport across cell membranes. In addition to the classical water transport function, peroxiporins have the unique capability to transport hydrogen peroxide (H2O2), a reactive oxygen species involved in various cellular signaling pathways and regulation of oxidative stress responses. The regulation of H2O2 levels is crucial for maintaining cellular homeostasis, and peroxiporins play a significant role in this process by modulating its intracellular and extracellular concentrations. This ability to facilitate the passage of H2O2 positions peroxiporins as key players in redox biology and cellular signaling, with implications for understanding and treating various diseases linked to oxidative stress and inflammation. This review provides updated information on the physiological roles of peroxiporins and their implications in disease, emphasizing their potential as novel biomarkers and drug targets in conditions where they are dysregulated, such as inflammation and cancer.

Clozapine is an antipsychotic drug whose accumulation in white cells can sometimes prove toxic; understanding the transporters and alleles responsible is thus highly desirable. We used a strategy in which a yeast (Saccharomyces cerevisiae) CRISPR-Cas9 knock-out library was exposed to cytotoxic concentrations of clozapine to determine those transporters whose absence made it more resistant; we also recognised the structural similarity of the fluorescent dye safranin O (also known as safranin T) to clozapine, allowing it to be used as a surrogate marker. Strains lacking the mitochondrial ABC transporter MDL1 (encoded by YLR188W) showed substantial resistance to clozapine. MDL1 overexpression also conferred extra sensitivity to clozapine and admitted a massive increase in the cellular and mitochondrial uptake of safranin O, as determined using flow cytometry and microscopically. Yeast lacking mitochondria showed no such unusual accumulation. Mitochondrial MDL1 is thus the main means of accumulation of clozapine in S. cerevisiae. The closest human homologue of S. cerevisiae MDL1 is ABCB10.

As microbial membranes are naturally impermeable to even the smallest biomolecules, transporter proteins are physiologically essential for normal cell functioning. This makes transporters a key target area for engineering enhanced cell factories. As part of the wider cellular transportome, aquaporins (AQPs) are responsible for transporting small polar solutes, encompassing many compounds which are of great interest for industrial biotechnology, including cell feedstocks, numerous commercially relevant polyols and even weak organic acids. In this review, examples of cell factory engineering by targeting AQPs are presented. These AQP modifications aid in redirecting carbon fluxes and boosting bioconversions either by enhanced feedstock uptake, improved intermediate retention, increasing product export into the media or superior cell viability against stressors with applications in both bacterial and yeast production platforms. Additionally, the future potential for AQP deployment and targeting is discussed, showcasing hurdles and considerations of this strategy as well as recent advances and future directions in the field. By leveraging the natural diversity of AQPs and breakthroughs in channel protein engineering, these transporters are poised to be promising tools capable of enhancing a wide variety of biotechnological processes.