Formation of a gated channel by a ligand-specific transport protein in the bacterial outer membrane.
Rutz J M,Liu J,Lyons J A,Goranson J,Armstrong S K,McIntosh M A,Feix J B,Klebba P E
Science (New York, N.Y.)
The ferric enterobactin receptor (FepA) is a high-affinity ligand-specific transport protein in the outer membrane of Gram-negative bacteria. Deletion of the cell-surface ligand-binding peptides of FepA generated mutant proteins that were incapable of high-affinity uptake but that instead formed nonspecific, passive channels in the outer membrane. Unlike native FepA, these pores acted independently of the accessory protein TonB, which suggests that FepA is a gated porin and that TonB acts as its gatekeeper by facilitating the entry of ligands into the FepA channel. The sequence homology among TonB-dependent proteins suggests that all ligand-specific outer membrane receptors may function by this gated-porin mechanism.
10.1126/science.1411544
Substitution of alanine for serine 250 in the murine fatty acid transport protein inhibits long chain fatty acid transport.
Stuhlsatz-Krouper S M,Bennett N E,Schaffer J E
The Journal of biological chemistry
The murine fatty acid transport protein (FATP) was identified on the basis of its ability to facilitate uptake of long chain fatty acids (LCFAs) when expressed in mammalian cells. To delineate FATP domains important for transport function, we cloned the human heart FATP ortholog. Comparison of the human, murine, and yeast amino acid sequences identified a highly conserved motif, IYTSGTTGXPK, also found in a number of proteins that form adenylated intermediates. We demonstrate that depletion of intracellular ATP dramatically reduces FATP-mediated LCFA uptake. Furthermore, wild-type FATP specifically binds [alpha-32P]azido-ATP. Introduction of a serine to alanine substitution (S250A) in the IYTSGTTGXPK motif produces an appropriately expressed and metabolized mutant FATP that demonstrates diminished LCFA transport function and decreased [alpha-32P]azido-ATP binding. These results are consistent with a mechanism of action for FATP involving ATP binding that is dependent on serine 250 of the IYTSGTTGXPK motif.
10.1074/jbc.273.44.28642
Lipid transfer proteins rectify inter-organelle flux and accurately deliver lipids at membrane contact sites.
Journal of lipid research
The endoplasmic reticulum (ER) is the main center for the synthesis of various lipid types in cells, and newly synthesized lipids are delivered from the ER to other organelles. In the past decade, various lipid transfer proteins (LTPs) have been recognized as mediators of lipid transport from the ER to other organelles; inter-organelle transport occurs at membrane contact sites (MCSs) and in a nonvesicular manner. Although the intermembrane transfer reaction catalyzed by LTPs is an equilibrium reaction, various types of newly synthesized lipids are transported unidirectionally in cells. This review provides a brief history of the inter-organelle trafficking of lipids and summarizes the structural and biochemical characteristics of the ceramide transport protein (CERT) as a typical LTP acting at MCSs. In addition, this review compares several LTP-mediated inter-organelle lipid trafficking systems and proposes that LTPs generate unidirectional fluxes of specific lipids between different organelles by indirect coupling with the metabolic reactions that occur in specific organelles. Moreover, the available data also suggest that the major advantage of LTP-mediated lipid transport at MCSs may be the accuracy of delivery. Finally, how cholesterol is enriched in the plasma membrane is discussed from a thermodynamic perspective.
10.1194/jlr.R085324
Ferritoid, a tissue-specific nuclear transport protein for ferritin in corneal epithelial cells.
Millholland John M,Fitch John M,Cai Cindy X,Gibney Eileen P,Beazley Kelly E,Linsenmayer Thomas F
The Journal of biological chemistry
Previously we reported that ferritin in corneal epithelial (CE) cells is a nuclear protein that protects DNA from UV damage. Since ferritin is normally cytoplasmic, in CE cells, a mechanism must exist that effects its nuclear localization. We have now determined that this involves a nuclear transport molecule we have termed ferritoid. Ferritoid is specific for CE cells and is developmentally regulated. Structurally, ferritoid contains multiple domains, including a functional SV40-type nuclear localization signal and a ferritin-like region of approximately 50% similarity to ferritin itself. This latter domain is likely responsible for the interaction between ferritoid and ferritin detected by co-immunoprecipitation analysis. To test functionally whether ferritoid is capable of transporting ferritin into the nucleus, we performed cotransfections of COS-1 cells with constructs for ferritoid and ferritin. Consistent with the proposed nuclear transport function for ferritoid, co-transfections with full-length constructs for ferritoid and ferritin resulted in a preferential nuclear localization of both molecules; this was not observed when the nuclear localization signal of ferritoid was deleted. Moreover, since ferritoid is structurally similar to ferritin, it may be an example of a nuclear transporter that evolved from the molecule it transports (ferritin).
10.1074/jbc.M210050200
Physiological response in E. coli to YdgR overexpression depends on whether the protein has an intact function.
Biochemical and biophysical research communications
Membrane transport proteins are essential for the transport of a wide variety of molecules across the cell membrane to maintain cellular homeostasis. Generally, these transport proteins can be overexpressed in a suitable host (bacteria, yeast, or mammalian cells), and it is well documented that overexpression of membrane proteins alters the global metabolomic and proteomic profiles of the host cells. In the present study, we investigated the physiological consequences of overexpression of a membrane transport protein YdgR that belongs to the POT/PTR family from E. coli by using the lab strain BL21 (DE3)pLysS in its functional and attenuated mutant YdgR-E33Q. We found significant differences between the omics (metabolomics and proteomics) profiles of the cells expressing functional YdgR as compared to cells expressing attenuated YdgR, e.g., upregulation of several uncharacterized y-proteins and enzymes involved in the metabolism of peptides and amino acids. Furthermore, molecular network analysis suggested a relatively higher presence of proline-containing tripeptides in cells expressing functional YdgR. We envisage that an in-depth investigation of physiological alterations due to protein over-expression may be used for the deorphanization of the y-gene transportome.
10.1016/j.bbrc.2023.04.032
Structural basis of outer dynein arm intraflagellar transport by the transport adaptor protein ODA16 and the intraflagellar transport protein IFT46.
Taschner Michael,Mourão André,Awasthi Mayanka,Basquin Jerome,Lorentzen Esben
The Journal of biological chemistry
Motile cilia are found on unicellular organisms such as the green alga , on sperm cells, and on cells that line the trachea and fallopian tubes in mammals. The motility of cilia relies on a number of large protein complexes including the force-generating outer dynein arms (ODAs). The transport of ODAs into cilia has been previously shown to require the transport adaptor ODA16, as well as the intraflagellar transport (IFT) protein IFT46, but the molecular mechanism by which ODAs are recognized and transported into motile cilia is still unclear. Here, we determined the high-resolution crystal structure of ODA16 (CrODA16) and mapped the binding to IFT46 and ODAs. The CrODA16 structure revealed a small 80-residue N-terminal domain and a C-terminal 8-bladed β-propeller domain that are both required for the association with the N-terminal 147 residues of IFT46. The dissociation constant of the IFT46-ODA16 complex was 200 nm, demonstrating that CrODA16 associates with the IFT complex with an affinity comparable with that of the individual IFT subunits. Furthermore, we show, using ODAs extracted from the axonemes of , that the C-terminal β-propeller but not the N-terminal domain of CrODA16 is required for the interaction with ODAs. These data allowed us to present an architectural model for ODA16-mediated IFT of ODAs.
10.1074/jbc.M117.780155
Ceramide Transport from the Endoplasmic Reticulum to the Trans Golgi Region at Organelle Membrane Contact Sites.
Hanada Kentaro
Advances in experimental medicine and biology
Lipids are the major constituents of all cell membranes and play dynamic roles in organelle structure and function. Although the spontaneous transfer of lipids between different membranes rarely occurs, lipids are appropriately transported between different organelles for their metabolism and to exert their functions in living cells. Proteins that have the biochemical capability to catalyze the intermembrane transfer of lipids are called lipid transfer proteins (LTPs). All organisms possess many types of LTPs. Recent studies revealed that LTPs are key players in the interorganelle transport of lipids at organelle membrane contact sites (MCSs). This chapter depicts how LTPs rationally operate at MCSs by using the ceramide transport protein CERT as a typical model for the LTP-mediated interorganelle transport of lipids.
10.1007/978-981-10-4567-7_5
Crystal structure of intraflagellar transport protein 80 reveals a homo-dimer required for ciliogenesis.
eLife
Oligomeric assemblies of intraflagellar transport (IFT) particles build cilia through sequential recruitment and transport of ciliary cargo proteins within cilia. Here we present the 1.8 Å resolution crystal structure of the IFT-B protein IFT80, which reveals the architecture of two N-terminal β-propellers followed by an α-helical extension. The N-terminal β-propeller tethers IFT80 to the IFT-B complex via IFT38 whereas the second β-propeller and the C-terminal α-helical extension result in IFT80 homo-dimerization. Using CRISPR/Cas to create biallelic frameshift mutations in IMCD3 mouse cells, we demonstrate that IFT80 is absolutely required for ciliogenesis. Structural mapping and rescue experiments reveal that human disease-causing missense mutations do not cluster within IFT80 and form functional IFT particles. Unlike missense mutant forms of IFT80, deletion of the C-terminal dimerization domain prevented rescue of ciliogenesis. Taken together our results may provide a first insight into higher order IFT complex formation likely required for IFT train formation.
10.7554/eLife.33067
Role of STARD4 and NPC1 in intracellular sterol transport.
Maxfield Frederick R,Iaea David B,Pipalia Nina H
Biochemistry and cell biology = Biochimie et biologie cellulaire
Cholesterol plays an important role in determining the biophysical properties of membranes in mammalian cells, and the concentration of cholesterol in membranes is tightly regulated. Cholesterol moves among membrane organelles by a combination of vesicular and nonvesicular transport pathways, but the details of these transport pathways are not well understood. In this review, we discuss the mechanisms for nonvesicular sterol transport with an emphasis on the role of STARD4, a small, soluble, cytoplasmic sterol transport protein. STARD4 can rapidly equilibrate sterol between membranes, especially membranes with anionic lipid headgroups. We also discuss the sterol transport in late endosomes and lysosomes, which is mediated by a soluble protein, NPC2, and a membrane protein, NPC1. Homozygous mutations in these proteins lead to a lysosomal lipid storage disorder, Niemann-Pick disease type C. Many of the disease-causing mutations in NPC1 are associated with degradation of the mutant NPC1 proteins in the endoplasmic reticulum. Several histone deacetylase inhibitors have been found to rescue the premature degradation of the mutant NPC1 proteins, and one of these is now in a small clinical trial.
10.1139/bcb-2015-0154
Transport protein trafficking in polarized cells.
Muth Theodore R,Caplan Michael J
Annual review of cell and developmental biology
In order to carry out their physiological functions, ion transport proteins must be targeted to the appropriate domains of cell membranes. Regulation of ion transport activity frequently involves the tightly controlled delivery of intracellular populations of transport proteins to the plasma membrane or the endocytic retrieval of transport proteins from the cell surface. Transport proteins carry signals embedded within their structures that specify their subcellular distributions and endow them with the capacity to participate in regulated membrane trafficking processes. Recently, a great deal has been learned about the biochemical nature of these signals, as well as about the cellular machinery that interprets them and acts upon their messages.
10.1146/annurev.cellbio.19.110701.161425