scholarly journals Dissection of the relative contribution of the Schizosaccharomyces pombe Ctr4 and Ctr5 proteins to the copper transport and cell surface delivery functions

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1021-1031 ◽  
Author(s):  
Jude Beaudoin ◽  
Dennis J. Thiele ◽  
Simon Labbé ◽  
Sergi Puig
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Schizosaccharomyces Pombe ◽  
Transmembrane Domain ◽  
Chimeric Proteins ◽  
High Affinity ◽  
Relative Contribution ◽  
Eukaryotic Organisms ◽  
Transport Complex ◽  
Cu Uptake

The Ctr1 family of proteins mediates high-affinity copper (Cu) acquisition in eukaryotic organisms. In the fission yeast Schizosaccharomyces pombe, Cu uptake is carried out by a heteromeric complex formed by the Ctr4 and Ctr5 proteins. Unlike human and Saccharomyces cerevisiae Ctr1 proteins, Ctr4 and Ctr5 are unable to function independently in Cu acquisition. Instead, both proteins physically interact with each other to form a Ctr4–Ctr5 heteromeric complex, and are interdependent for secretion to the plasma membrane and Cu transport activity. In this study, we used S. cerevisiae mutants that are defective in high-affinity Cu uptake to dissect the relative contribution of Ctr4 and Ctr5 to the Cu transport function. Functional complementation and localization assays show that the conserved Met-X3-Met motif in transmembrane domain 2 of the Ctr5 protein is dispensable for the functionality of the Ctr4–Ctr5 complex, whereas the Met-X3-Met motif in the Ctr4 protein is essential for function and for localization of the hetero-complex to the plasma membrane. Moreover, Ctr4/Ctr5 chimeric proteins reveal unique properties found either in Ctr4 or in Ctr5, and are sufficient for Cu uptake on the cell surface of Sch. pombe cells. Functional chimeras contain the Ctr4 central and Ctr5 carboxyl-terminal domains (CTDs). We propose that the Ctr4 central domain mediates Cu transport in this hetero-complex, whereas the Ctr5 CTD functions in the regulation of trafficking of the Cu transport complex to the cell surface.

scholarly journals Amiloride Uptake and Toxicity in Fission Yeast Are Caused by the Pyridoxine Transporter Encoded by bsu1+ (car1+)

2005 ◽  
Vol 4 (2) ◽  
pp. 319-326 ◽  
Author(s):  
Jürgen Stolz ◽  
Heike J. P. Wöhrmann ◽  
Christian Vogl
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Multidrug Resistance ◽  
Schizosaccharomyces Pombe ◽  
Fission Yeast ◽  
Vitamin B ◽  
Acidic Ph ◽  
Ph Optimum ◽  
Sodium Transporters ◽  
High Affinity

ABSTRACT Amiloride, a diuretic drug that acts by inhibition of various sodium transporters, is toxic to the fission yeast Schizosaccharomyces pombe. Previous work has established that amiloride sensitivity is caused by expression of car1 +, which encodes a protein with similarity to plasma membrane drug/proton antiporters from the multidrug resistance family. Here we isolated car1 + by complementation of Saccharomyces cerevisiae mutants that are deficient in pyridoxine biosynthesis and uptake. Our data show that Car1p represents a new high-affinity, plasma membrane-localized import carrier for pyridoxine, pyridoxal, and pyridoxamine. We therefore propose the gene name bsu1 + (for vitamin B6 uptake) to replace car1 +. Bsu1p displays an acidic pH optimum and is inhibited by various protonophores, demonstrating that the protein works as a proton symporter. The expression of bsu1 + is associated with amiloride sensitivity and pyridoxine uptake in both S. cerevisiae and S. pombe cells. Moreover, amiloride acts as a competitor of pyridoxine uptake, demonstrating that both compounds are substrates of Bsu1p. Taken together, our data show that S. pombe and S. cerevisiae possess unrelated plasma membrane pyridoxine transporters. The S. pombe protein may be structurally related to the unknown human pyridoxine transporter, which is also inhibited by amiloride.

scholarly journals Truncated isoforms inhibit [3H]prazosin binding and cellular trafficking of native human α1A-adrenoceptors

1999 ◽  
Vol 343 (1) ◽  
pp. 231-239 ◽  
Author(s):  
Francis COGÉ ◽  
Sophie-Pénélope GUENIN ◽  
Anne RENOUARD-TRY ◽  
Hervé RIQUE ◽  
Christine OUVRY ◽  
...  
Keyword(s):  
Cell Surface ◽  
Human Liver ◽  
Human Heart ◽  
Transmembrane Domain ◽  
Splice Variants ◽  
Biological Properties ◽  
Transfected Cells ◽  
High Affinity ◽  
Intracellular Compartments ◽  
Truncated Isoforms

We have identified from human liver eight α1A-adrenoceptor (α1A-AR) splice variants that were also expressed in human heart, prostate and hippocampus. Three of these α1A-AR isoforms (α1A-1-AR, α1A-2a-AR and α1A-3a-AR) gave rise to receptors with seven transmembrane domains (7TMα1A-AR). The other five (α1A-2b-AR, α1A-2c-AR, α1A-3c-AR, α1A-5-AR and α1A-6-AR) led to truncated receptors lacking transmembrane domain VII (6TMα1A-AR). The 7TMα1A-AR isoforms transiently expressed in COS-7 cells bound [3H]prazosin with high affinity (Kd 0.2 nM) and mediated a noradrenaline (norepinephrine)-induced increase in cytoplasmic free Ca2+ concentration, whereas the 6TMα1A-AR isoforms were incapable of ligand binding and signal transduction. Immunocytochemical studies with N-terminal epitope-tagged α1A-AR isoforms showed that the 7TMα1A-AR isoforms were present both at the cell surface and in intracellular compartments, whereas the 6TMα1A-AR isoforms were exclusively localized within the cell. Interestingly, in co-transfected cells, each truncated α1A-AR isoform inhibited [3H]prazosin binding and cell-surface trafficking of the co-expressed ‘original’ 7TMα1A-1-AR. However, there was no modification of either the [3H]prazosin-binding affinity or the pharmacological properties of α1A-1-AR. Immunoblotting experiments revealed that co-expression of the α1A-1-AR with 6TMα1A-AR isoforms did not impair α1A-1-AR expression. Therefore the expression in human tissues of many truncated isoforms constitutes a new regulation pathway of biological properties of α1A-AR.

scholarly journals Human macrophage colony-stimulating factor is expressed at and shed from the cell surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2182-2188 ◽  
Author(s):  
DP Tuck ◽  
DP Cerretti ◽  
A Hand ◽  
A Guha ◽  
S Sorba ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Transmembrane Domain ◽  
Surface Membrane ◽  
Mutant Form ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.

scholarly journals Specific Retrieval of the Exocytic SNARE Snc1p from Early Yeast Endosomes

2000 ◽  
Vol 11 (1) ◽  
pp. 23-38 ◽  
Author(s):  
Michael J. Lewis ◽  
Benjamin J. Nichols ◽  
Cristina Prescianotto-Baschong ◽  
Howard Riezman ◽  
Hugh R. B. Pelham
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Golgi Apparatus ◽  
Cell Surface ◽  
Immunoelectron Microscopy ◽  
Fluorescent Protein ◽  
Transmembrane Domain ◽  
Early Endosomes ◽  
Internal Structures ◽  
Green Fluorescent

Many endocytosed proteins in yeast travel to the vacuole, but some are recycled to the plasma membrane. We have investigated the recycling of chimeras containing green fluorescent protein (GFP) and the exocytic SNARE Snc1p. GFP-Snc1p moves from the cell surface to internal structures when Golgi function or exocytosis is blocked, suggesting continuous recycling via the Golgi. Internalization is mediated by a conserved cytoplasmic signal, whereas diversion from the vacuolar pathway requires sequences within and adjacent to the transmembrane domain. Delivery from the Golgi to the surface is also influenced by the transmembrane domain, but the requirements are much less specific. Recycling requires the syntaxins Tlg1p and Tlg2p but not Pep12p or proteins such as Vps4p and Vps5p that have been implicated in late endosome–Golgi traffic. Subtle changes to the recycling signal cause GFP-Snc1p to accumulate preferentially in punctate internal structures, although it continues to recycle to the surface. The internal GFP-Snc1p colocalizes with Tlg1p, and immunofluorescence and immunoelectron microscopy reveal structures that contain Tlg1p, Tlg2p, and Kex2p but lack Pep12p and Sec7p. We propose that these represent early endosomes in which sorting of Snc1p and late Golgi proteins occurs, and that transport can occur directly from them to the Golgi apparatus.

scholarly journals Human macrophage colony-stimulating factor is expressed at and shed from the cell surface

Blood ◽  
1994 ◽  
Vol 84 (7) ◽  
pp. 2182-2188 ◽  
Author(s):  
DP Tuck ◽  
DP Cerretti ◽  
A Hand ◽  
A Guha ◽  
S Sorba ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Transmembrane Domain ◽  
Surface Membrane ◽  
Mutant Form ◽  
Colony Stimulating Factor ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.

scholarly journals Role of the transmembrane and extracytoplasmic domain of beta subunits in subunit assembly, intracellular transport, and functional expression of Na,K-pumps.

1993 ◽  
Vol 123 (6) ◽  
pp. 1751-1759 ◽  
Author(s):  
P Jaunin ◽  
F Jaisser ◽  
A T Beggah ◽  
K Takeyasu ◽  
P Mangeat ◽  
...  
Keyword(s):  
Cell Surface ◽  
Intracellular Transport ◽  
Transmembrane Domain ◽  
Functional Expression ◽  
Beta Subunit ◽  
Beta Subunits ◽  
Chimeric Proteins ◽  
Er Retention ◽  
Retention Signal

The ubiquitous Na,K- and the gastric H,K-pumps are heterodimeric plasma membrane proteins composed of an alpha and a beta subunit. The H,K-ATPase beta subunit (beta HK) can partially act as a surrogate for the Na,K-ATPase beta subunit (beta NK) in the formation of functional Na,K-pumps (Horisberger et al., 1991. J. Biol. Chem. 257:10338-10343). We have examined the role of the transmembrane and/or the ectodomain of beta NK in (a) its ER retention in the absence of concomitant synthesis of Na,K-ATPase alpha subunits (alpha NK) and (b) the functional expression of Na,K-pumps at the cell surface and their activation by external K+. We have constructed chimeric proteins between Xenopus beta NK and rabbit beta HK by exchanging their NH2-terminal plus transmembrane domain with their COOH-terminal ectodomain (beta NK/HK, beta HK/NK). We have expressed these constructs with or without coexpression of alpha NK in the Xenopus oocyte. In the absence of alpha NK, Xenopus beta NK and all chimera that contained the ectodomain of beta NK were retained in the ER while beta HK and all chimera with the ectodomain of beta HK could leave the ER suggesting that ER retention of unassembled Xenopus beta NK is mediated by a retention signal in the ectodomain. When coexpressed with alpha NK, only beta NK and beta NK/HK chimera assembled efficiently with alpha NK leading to similar high expression of functional Na,K-pumps at the cell surface that exhibited, however, a different apparent K+ affinity. beta HK or chimera with the transmembrane domain of beta HK assembled less efficiently with alpha NK leading to lower expression of functional Na,K-pumps with a different apparent K+ affinity. The data indicate that the transmembrane domain of beta NK is important for efficient assembly with alpha NK and that both the transmembrane and the ectodomain of beta subunits play a role in modulating the transport activity of Na,K-pumps.

scholarly journals Dual Loss of ER Export and Endocytic Signals with Altered Melanosome Morphology in the silver Mutation of Pmel17

2006 ◽  
Vol 17 (8) ◽  
pp. 3598-3612 ◽  
Author(s):  
Alexander C. Theos ◽  
Joanne F. Berson ◽  
Sarah C. Theos ◽  
Kathryn E. Herman ◽  
Dawn C. Harper ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Pigment Cell ◽  
Integral Membrane Protein ◽  
Chimeric Proteins ◽  
Combined Effects ◽  
Er Export ◽  
Endocytic Compartments

Pmel17 is a pigment cell-specific integral membrane protein that participates in the formation of the intralumenal fibrils upon which melanins are deposited in melanosomes. The Pmel17 cytoplasmic domain is truncated by the mouse silver mutation, which is associated with coat hypopigmentation in certain strain backgrounds. Here, we show that the truncation interferes with at least two steps in Pmel17 intracellular transport, resulting in defects in melanosome biogenesis. Human Pmel17 engineered with the truncation found in the mouse silver mutant (hPmel17si) is inefficiently exported from the endoplasmic reticulum (ER). Localization and metabolic pulse-chase analyses with site-directed mutants and chimeric proteins show that this effect is due to the loss of a conserved C-terminal valine that serves as an ER exit signal. hPmel17si that exits the ER accumulates abnormally at the plasma membrane due to the loss of a di-leucine–based endocytic signal. The combined effects of reduced ER export and endocytosis significantly deplete Pmel17 within endocytic compartments and delay proteolytic maturation required for premelanosome-like fibrillogenesis. The ER export delay and cell surface retention are also observed for endogenous Pmel17si in melanocytes from silver mice, within which Pmel17 accumulation in premelanosomes is dramatically reduced. Mature melanosomes in these cells are larger, rounder, more highly pigmented, and less striated than in control melanocytes. These data reveal a dual sorting defect in a natural mutant of Pmel17 and support a requirement of endocytic trafficking in Pmel17 fibril formation.

scholarly journals Structural Domains of the Herpes Simplex Type 1 gD Protein that Restrict HIV-1 Particle Infectivity

2021 ◽  
Author(s):  
Sachith Polpitiya Arachchige ◽  
Wyatt Henke ◽  
Maria Kalamvoki ◽  
Edward B. Stephens
Keyword(s):  
Cell Surface ◽  
Transmembrane Domain ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Chimeric Proteins ◽  
Structural Domains ◽  
Virus Particles ◽  
Hiv 1 ◽  
Hsv 1

Previously, we showed that the presence of the herpes simplex virus type 1 (HSV-1) gD glycoprotein but not gB potently restricted HIV-1 particle infectivity. This restriction was characterized by incorporation of HSV-1 gD and the exclusion of the HIV-1 gp120/gp41 from budding virus particles. To determine the structural domains involved in gD restriction of HIV-1, a series of deletion mutants and chimeric proteins between gD and the non-restrictive gB were generated. Our results show that deletion of the cytoplasmic tail domain (CTD) of gD or that replacement of the transmembrane domain (TMD) with the TMD from gB slightly reduced restriction activity. However, replacement of the gD CTD with that of gB resulted in lower cell surface expression, significantly less incorporation into HIV-1 particles, and inefficient restriction of the release of infectious HIV-1. Analysis of gB/gD chimeric proteins revealed that removal of the gB CTD or replacement with gD CTD resulted in enhanced surface expression and an increase in restriction activity. Finally, we show that expression of gD without other HSV-1 proteins resulted in gD fractionation into detergent resistant membranes (DRM) and that gD co-localized with the raft marker GM1, which may partially explain its incorporation into budding virus particles. Taken together, our results suggest that expression of gD at the cell surface is likely a major factor but that other intrinsic properties are also involved in the gD-mediated restriction of HIV-1 particle infectivity. IMPORTANCE Previously, we showed that unlike the HSV-1, the presence of the gD glycoprotein in virus producer cells but not gB potently restricted HIV-1 particle infectivity. To better understand the relationship between cell surface expression, virus incorporation and restriction of HIV-1, we analyzed a series of deletion mutants and chimeric proteins in which domains of gD and gB were swapped. Our results indicate that: a) gD/gB chimeras having the cytoplasmic domain (CTD) of gB significantly reduced cell surface expression, release from cells, incorporation into virus, and reduced HIV-1 restriction; b) removal of the gB CTD or replacement with the gD CTD resulted in better surface expression, incorporation into HIV-1, and enhanced restriction; and c) the transmembrane domain of gB can influence transport and ultimately effect incorporation of gB into HIV-1. Overall, these data support a role for gD surface expression as crucial to restriction of infectious HIV-1 release.

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