scholarly journals Functional studies of hephaestin in yeast: evidence for multicopper oxidase activity in the endocytic pathway

2003 ◽  
Vol 375 (3) ◽  
pp. 793-798 ◽  
Author(s):  
Liangtao LI ◽  
Chris D. VULPE ◽  
Jerry KAPLAN
Keyword(s):  
Cell Surface ◽  
Oxidase Activity ◽  
Transmembrane Domain ◽  
Multicopper Oxidase ◽  
Full Length ◽  
Endocytic Pathway ◽  
Functional Studies ◽  
Dependent Protein ◽  
A Cell ◽  
Growth Phenotype

Hephaestin is a mammalian gene that encodes a predicted multicopper oxidase required for intestinal iron export. To examine if hephaestin can act as a ferroxidase, we studied yeast strains transformed with plasmids containing both a full-length hephaestin and a hephaestin lacking a transmembrane domain. Yeast with a deletion in FET3, which encodes a cell-surface multicopper oxidase, cannot grow on low-iron media. Expression of full-length hephaestin could complement the low-iron growth phenotype of a Δfet3 strain. Complementation of Δfet3 cells by hephaestin required genes that encode proteins necessary for the copper loading of Fet3p, including CCC2 and GEF1. Expression of hephaestin in Δfet3 cells led to an increase in both iron transport and oxidase activity. These results demonstrate that hephaestin is a copper-dependent protein. In contrast with Fet3p, which is found on the cell surface, hephaestin was co-localized with Pep12p-containing vesicles. Inhibition of endocytosis or deletion of both the vacuolar iron transporters (SMF3 and FET5/FTH1) prevented hephaestin from complementing the low-iron growth phenotype of Δfet3 cells, suggesting that hephaestin is functioning within the endocytic apparatus.

scholarly journals A knockout cell library of GPI biosynthetic genes for functional studies of GPI-anchored proteins

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Si-Si Liu ◽  
Yi-Shi Liu ◽  
Xin-Yu Guo ◽  
Yoshiko Murakami ◽  
Ganglong Yang ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Trafficking ◽  
Mammalian Cells ◽  
Surface Expression ◽  
Human Embryonic Kidney ◽  
Functional Studies ◽  
Cell Library ◽  
Biological Phenomena ◽  
293 Cells ◽  
A Cell

AbstractOver 100 kinds of proteins are expressed as glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) on the cell surface in mammalian cells. GPI-APs possess unique properties in terms of their intracellular trafficking and association with lipid rafts. Although it is clear that GPI-APs play critical roles in various biological phenomena, it is poorly understood how the GPI moiety contributes to these mechanisms. More than 30 genes are involved in the correct biosynthesis of GPI-APs. We here constructed a cell library in which 32 genes involved in GPI biosynthesis were knocked out in human embryonic kidney 293 cells. Using the cell library, the surface expression and sensitivity to phosphatidylinositol-specific phospholipase C of GPI-APs were analyzed. Furthermore, we identified structural motifs of GPIs that are recognized by a GPI-binding toxin, aerolysin. The cell-based GPI-knockout library could be applied not only to basic researches, but also to applications and methodologies related to GPI-APs.

scholarly journals Structures and an activation mechanism of human potassium-chloride cotransporters

2020 ◽  
Vol 6 (50) ◽  
pp. eabc5883 ◽  
Author(s):  
Yuan Xie ◽  
Shenghai Chang ◽  
Cheng Zhao ◽  
Feng Wang ◽  
Si Liu ◽  
...  
Keyword(s):  
Potassium Chloride ◽  
Volume Regulation ◽  
Transmembrane Domain ◽  
Cell Volume Regulation ◽  
Full Length ◽  
Aminobutyric Acid ◽  
Activation Mechanism ◽  
Inhibitory Peptide ◽  
Domain Swap ◽  
Functional Studies

Potassium-chloride cotransporters KCC1 to KCC4 mediate the coupled export of potassium and chloride across the plasma membrane and play important roles in cell volume regulation, auditory system function, and γ-aminobutyric acid (GABA) and glycine-mediated inhibitory neurotransmission. Here, we present 2.9- to 3.6-Å resolution structures of full-length human KCC2, KCC3, and KCC4. All three KCCs adopt a similar overall architecture, a domain-swap dimeric assembly, and an inward-facing conformation. The structural and functional studies reveal that one unexpected N-terminal peptide binds at the cytosolic facing cavity and locks KCC2 and KCC4 at an autoinhibition state. The C-terminal domain (CTD) directly interacts with the N-terminal inhibitory peptide, and the relative motions between the CTD and the transmembrane domain (TMD) suggest that CTD regulates KCCs’ activities by adjusting the autoinhibitory effect. These structures provide the first glimpse of full-length structures of KCCs and an autoinhibition mechanism among the amino acid–polyamine-organocation transporter superfamily.

scholarly journals Delineation of the endocytic pathway of substance P and its seven-transmembrane domain NK1 receptor.

1995 ◽  
Vol 6 (5) ◽  
pp. 509-524 ◽  
Author(s):  
E F Grady ◽  
A M Garland ◽  
P D Gamp ◽  
M Lovett ◽  
D G Payan ◽  
...  
Keyword(s):  
Substance P ◽  
Cell Surface ◽  
Transmembrane Domain ◽  
Endocytic Pathway ◽  
Atpase Inhibitor ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Endosomal Acidification ◽  
Neurokinin 1 ◽  
New Protein

Many of the actions of the neuropeptide substance P (SP) that are mediated by the neurokinin 1 receptor (NK1-R) desensitize and resensitize, which may be associated with NK1-R endocytosis and recycling. We delineated this endocytic pathway in transfected cells by confocal microscopy using cyanine 3-SP and NK1-R antibodies. SP and the NK1-R were internalized into the same clathrin immunoreactive vesicles, and then sorted into different compartments. The NK1-R was colocalized with a marker of early endosomes, but not with markers of late endosomes or lysosomes. We quantified the NK1-R at the cell surface by incubating cells with an antibody to an extracellular epitope. After exposure to SP, there was a loss and subsequent recovery of surface NK1-R. The loss was prevented by hypertonic sucrose and potassium depletion, inhibitors of clathrin-mediated endocytosis. Recovery was independent of new protein synthesis because it was unaffected by cycloheximide. Recovery required endosomal acidification because it was prevented by an H(+)-ATPase inhibitor. The fate of internalized 125I-SP was examined by chromatography. SP was intact at the cell surface and in early endosomes, but slowly degraded in perinuclear vesicles. We conclude that SP induces clathrin-dependent internalization of the NK1-R. The SP/NK1-R complex dissociates in acidified endosomes. SP is degraded, whereas the NK1-R recycles to the cell surface.

scholarly journals NKG2D Ligand MICA Is Retained in the cis-Golgi Apparatus by Human Cytomegalovirus Protein UL142

2009 ◽  
Vol 83 (23) ◽  
pp. 12345-12354 ◽  
Author(s):  
Omodele Ashiru ◽  
Neil J. Bennett ◽  
Louise H. Boyle ◽  
Mair Thomas ◽  
John Trowsdale ◽  
...  
Keyword(s):  
Golgi Apparatus ◽  
Cell Surface ◽  
Nk Cells ◽  
Mhc Class I ◽  
Human Cytomegalovirus ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Full Length ◽  
Class I ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) evades T-cell recognition by down-regulating expression of major histocompatibility complex (MHC) class I and II molecules on the surfaces of infected cells. Contrary to the “missing-self” hypothesis, HCMV-infected cells are refractory to lysis by natural killer (NK) cells. Inhibition of NK cell function is mediated by a number of HCMV immune evasion molecules, which operate by delivering inhibitory signals to NK cells and preventing engagement of activating ligands. One such molecule is UL142, which is an MHC class I-related glycoprotein encoded by clinical isolates and low-passage-number strains of HCMV. UL142 is known to down-modulate surface expression of MHC class I-related chain A (MICA), which is a ligand of the activating NK receptor NKG2D. However, the mechanism by which UL142 interferes with MICA is unknown. Here, we show that UL142 localizes predominantly to the endoplasmic reticulum (ER) and cis-Golgi apparatus. The transmembrane domain of UL142 mediates its ER localization, while we propose that the UL142 luminal domain is involved in its cis-Golgi localization. We also confirm that UL142 down-modulates surface expression of full-length MICA alleles while having no effect on the truncated allele MICA*008. However, we demonstrate for the first time that UL142 retains full-length MICA alleles in the cis-Golgi apparatus. In addition, we propose that UL142 interacts with nascent MICA en route to the cell surface but not mature MICA at the cell surface. Our data also demonstrate that the UL142 luminal and transmembrane domains are involved in recognition and intracellular sequestration of full-length MICA alleles.

scholarly journals Leukocytes with chromosome Y loss have reduced abundance of the cell surface immunoprotein CD99

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Jonas Mattisson ◽  
Marcus Danielsson ◽  
Maria Hammond ◽  
Hanna Davies ◽  
Caroline J. Gallant ◽  
...  
Keyword(s):  
Cell Surface ◽  
Single Cells ◽  
Surface Protein ◽  
Protein Abundance ◽  
Blood Leukocytes ◽  
Chromosome Y ◽  
Increased Risk ◽  
A Cell ◽  
Pseudoautosomal Regions ◽  
Male Specific

AbstractMosaic loss of chromosome Y (LOY) in immune cells is a male-specific mutation associated with increased risk for morbidity and mortality. The CD99 gene, positioned in the pseudoautosomal regions of chromosomes X and Y, encodes a cell surface protein essential for several key properties of leukocytes and immune system functions. Here we used CITE-seq for simultaneous quantification of CD99 derived mRNA and cell surface CD99 protein abundance in relation to LOY in single cells. The abundance of CD99 molecules was lower on the surfaces of LOY cells compared with cells without this aneuploidy in all six types of leukocytes studied, while the abundance of CD proteins encoded by genes located on autosomal chromosomes were independent from LOY. These results connect LOY in single cells with immune related cellular properties at the protein level, providing mechanistic insight regarding disease vulnerability in men affected with mosaic chromosome Y loss in blood leukocytes.

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