scholarly journals Control of Protein and Sterol Trafficking by Antagonistic Activities of a Type IV P-type ATPase and Oxysterol Binding Protein Homologue

2009 ◽  
Vol 20 (12) ◽  
pp. 2920-2931 ◽  
Author(s):  
Baby-Periyanayaki Muthusamy ◽  
Sumana Raychaudhuri ◽  
Paramasivam Natarajan ◽  
Fumiyoshi Abe ◽  
Ke Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Binding Protein ◽  
Feedback Mechanism ◽  
Family Type ◽  
Growth Defect ◽  
Oxysterol Binding Protein ◽  
Type Iv ◽  
P Type

The oxysterol binding protein homologue Kes1p has been implicated in nonvesicular sterol transport in Saccharomyces cerevisiae. Kes1p also represses formation of protein transport vesicles from the trans-Golgi network (TGN) through an unknown mechanism. Here, we show that potential phospholipid translocases in the Drs2/Dnf family (type IV P-type ATPases [P4-ATPases]) are downstream targets of Kes1p repression. Disruption of KES1 suppresses the cold-sensitive (cs) growth defect of drs2Δ, which correlates with an enhanced ability of Dnf P4-ATPases to functionally substitute for Drs2p. Loss of Kes1p also suppresses a drs2-ts allele in a strain deficient for Dnf P4-ATPases, suggesting that Kes1p antagonizes Drs2p activity in vivo. Indeed, Drs2-dependent phosphatidylserine translocase (flippase) activity is hyperactive in TGN membranes from kes1Δ cells and is potently attenuated by addition of recombinant Kes1p. Surprisingly, Drs2p also antagonizes Kes1p activity in vivo. Drs2p deficiency causes a markedly increased rate of cholesterol transport from the plasma membrane to the endoplasmic reticulum (ER) and redistribution of endogenous ergosterol to intracellular membranes, phenotypes that are Kes1p dependent. These data suggest a homeostatic feedback mechanism in which appropriately regulated flippase activity in the Golgi complex helps establish a plasma membrane phospholipid organization that resists sterol extraction by a sterol binding protein.

scholarly journals Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

2009 ◽  
Vol 20 (5) ◽  
pp. 1388-1399 ◽  
Author(s):  
Mike Ngo ◽  
Neale D. Ridgway
Keyword(s):  
Secretory Pathway ◽  
Protein Transport ◽  
Binding Protein ◽  
Dominant Negative ◽  
Ph Domain ◽  
Oxysterol Binding Protein ◽  
Large Gene ◽  
Dominant Negative Variant

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.

Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule

1993 ◽  
Vol 264 (2) ◽  
pp. C302-C310 ◽  
Author(s):  
H. Birn ◽  
J. Selhub ◽  
E. I. Christensen
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Brush Border ◽  
Intracellular Transport ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Binding Protein ◽  
Rat Kidney ◽  
Folate Binding Protein

Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.

scholarly journals Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

2010 ◽  
Vol 21 (13) ◽  
pp. 2327-2337 ◽  
Author(s):  
Sokha Nhek ◽  
Mike Ngo ◽  
Xuemei Yang ◽  
Michelle M. Ng ◽  
Seth J. Field ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Binding Protein ◽  
Critical Role ◽  
Protein Kinase D ◽  
Cholesterol Depletion ◽  
Oxysterol Binding Protein ◽  
Golgi Localization ◽  
Golgi Network

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.

Sterol trafficking between the endoplasmic reticulum and plasma membrane in yeast

2006 ◽  
Vol 34 (3) ◽  
pp. 356-358 ◽  
Author(s):  
D.P. Sullivan ◽  
H. Ohvo-Rekilä ◽  
N.A. Baumann ◽  
C.T. Beh ◽  
A.K. Menon
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Wild Type Strain ◽  
Binding Pocket ◽  
Oxysterol Binding Protein ◽  
Sterol Transport ◽  
Transport Cycle ◽  
Independent Mechanism

We recently showed that transport of ergosterol from the ER (endoplasmic reticulum) to the sterol-enriched PM (plasma membrane) in yeast occurs by a non-vesicular (Sec18p-independent) mechanism that results in the equilibration of sterol pools in the two organelles [Baumann, Sullivan, Ohvo-Rekilä, Simonot, Pottekat, Klaassen, Beh and Menon (2005) Biochemistry 44, 5816–5826]. To explore how this occurs, we tested the role of proteins that might act as sterol transporters. We chose to study oxysterol-binding protein homologues (Osh proteins), a family of seven proteins in yeast, all of which contain a putative sterol-binding pocket. Recent structural analyses of one of the Osh proteins [Im, Raychaudhuri, Prinz and Hurley (2005) Nature (London) 437, 154–158] suggested a possible transport cycle in which Osh proteins could act to equilibrate ER and PM pools of sterol. Our results indicate that the transport of newly synthesized ergosterol from the ER to the PM in an OSH deletion mutant lacking all seven Osh proteins is slowed only 5-fold relative to the isogenic wild-type strain. Our results suggest that the Osh proteins are not sterol transporters themselves, but affect sterol transport in vivo indirectly by affecting the ability of the PM to sequester sterols.

scholarly journals The Vitamin D Receptor Is Present in Caveolae-Enriched Plasma Membranes and Binds 1α,25(OH)2-Vitamin D3in Vivo and in Vitro

2004 ◽  
Vol 18 (11) ◽  
pp. 2660-2671 ◽  
Author(s):  
Johanna A. Huhtakangas ◽  
Christopher J. Olivera ◽  
June E. Bishop ◽  
Laura P. Zanello ◽  
Anthony W. Norman
Keyword(s):  
Vitamin D ◽  
Plasma Membrane ◽  
Vitamin D Receptor ◽  
Plasma Membranes ◽  
Binding Protein ◽  
Kidney Tissue ◽  
Mouse Lung ◽  
Nuclear Fraction

Abstract The steroid hormone 1α,25(OH)2-vitamin D3 (1,25D) regulates gene transcription through a nuclear receptor [vitamin D receptor (VDR)] and initiation of rapid cellular responses through a putative plasma membrane-associated receptor (VDRmem). This study characterized the VDRmem present in a caveolae-enriched membrane fraction (CMF), a site of accumulation of signal transduction agents. Saturable and specific [3H]-1,25D binding in vitro was found in CMF of chick, rat, and mouse intestine; mouse lung and kidney; and human NB4 leukemia and rat ROS 17/2.8 osteoblast-like cells; in all cases the 1,25D KD binding dissociation constant = 1–3 nm. Our data collectively support the classical VDR being the VDRmem in caveolae: 1) VDR antibody immunoreactivity was detected in CMF of all tissues tested; 2) competitive binding of [3H]-1,25D by eight analogs of 1,25D was significantly correlated between nuclei and CMF (r2 = 0.95) but not between vitamin D binding protein (has a different ligand binding specificity) and CMF; 3) confocal immunofluorescence microscopy of ROS 17/2.8 cells showed VDR in close association with the caveolae marker protein, caveolin-1, in the plasma membrane region; 4) in vivo 1,25D pretreatment reduced in vitro [3H]-1,25D binding by 30% in chick and rat intestinal CMF demonstrating in vivo occupancy of the CMF receptor by 1,25D; and 5) comparison of [3H]-1,25D binding in VDR KO and WT mouse kidney tissue showed 85% reduction in VDR KO CMF and 95% reduction in VDR KO nuclear fraction. This study supports the presence of VDR as the 1,25D-binding protein associated with plasma membrane caveolae.

scholarly journals Yeast P4-ATPases Drs2p and Dnf1p Are Essential Cargos of the NPFXD/Sla1p Endocytic Pathway

2007 ◽  
Vol 18 (2) ◽  
pp. 487-500 ◽  
Author(s):  
Ke Liu ◽  
Zhaolin Hua ◽  
Joshua A. Nepute ◽  
Todd R. Graham
Keyword(s):  
Plasma Membrane ◽  
Protein Transport ◽  
Endocytic Pathway ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Sensitive Mutant ◽  
C Terminus ◽  
P Type ◽  
Golgi Network ◽  
Endocytic Pathways

Drs2p family P-type ATPases (P4-ATPases) are required in multiple vesicle-mediated protein transport steps and are proposed to be phospholipid translocases (flippases). The P4-ATPases Drs2p and Dnf1p cycle between the exocytic and endocytic pathways, and here we define endocytosis signals required by these proteins to maintain a steady-state localization to internal organelles. Internalization of Dnf1p from the plasma membrane uses an NPFXD endocytosis signal and its recognition by Sla1p, part of an endocytic coat/adaptor complex with clathrin, Pan1p, Sla2p/End4p, and End3p. Drs2p has multiple endocytosis signals, including two NPFXDs near the C terminus and PEST-like sequences near the N terminus that may mediate ubiquitin (Ub)-dependent endocytosis. Drs2p localizes to the trans-Golgi network in wild-type cells and accumulates on the plasma membrane when both the Ub- and NPFXD-dependent endocytic mechanisms are inactivated. Surprisingly, the pan1-20 temperature-sensitive mutant is constitutively defective for Ub-dependent endocytosis but is not defective for NPFXD-dependent endocytosis at the permissive growth temperature. To sustain viability of pan1-20, Drs2p must be endocytosed through the NPFXD/Sla1p pathway. Thus, Drs2p is an essential endocytic cargo in cells compromised for Ub-dependent endocytosis. These results demonstrate an essential role for endocytosis in retrieving proteins back to the Golgi, and they define critical cargos of the NPFXD/Sla1p system.

scholarly journals In vivo cross-linking supports a head-to-tail mechanism for regulation of the plant plasma membrane P-type H+-ATPase

2018 ◽  
Vol 293 (44) ◽  
pp. 17095-17106 ◽  
Author(s):  
Thao T. Nguyen ◽  
Grzegorz Sabat ◽  
Michael R. Sussman
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Unnatural Amino Acid ◽  
Proton Pumping ◽  
Intramolecular Interactions ◽  
Cross Linking ◽  
Regulatory Domain ◽  
P Type ◽  
Plant Plasma Membrane

In higher plants, a P-type proton-pumping ATPase generates the proton-motive force essential for the function of all other transporters and for proper growth and development. X-ray crystallographic studies of the plant plasma membrane proton pump have provided information on amino acids involved in ATP catalysis but provided no information on the structure of the C-terminal regulatory domain. Despite progress in elucidating enzymes involved in the signaling pathways that activate or inhibit this pump, the site of interaction of the C-terminal regulatory domain with the catalytic domains remains a mystery. Genetic studies have pointed to amino acids in various parts of the protein that may be involved, but direct chemical evidence for which ones are specifically interacting with the C terminus is lacking. In this study, we used in vivo cross-linking experiments with a photoreactive unnatural amino acid, p-benzoylphenylalanine, and tandem MS to obtain direct evidence that the C-terminal regulatory domain interacts with amino acids located within the N-terminal actuator domain. Our observations are consistent with a mechanism in which intermolecular, rather than intramolecular, interactions are involved. Our model invokes a “head-to-tail” organization of ATPase monomers in which the C-terminal domain of one ATPase molecule interacts with the actuator domain of another ATPase molecule. This model serves to explain why cross-linked peptides are found only in dimers and trimers, and it is consistent with prior studies suggesting that within the membrane the protein can be organized as homopolymers, including dimers, trimers, and hexamers.

scholarly journals Analysis of oxysterol binding protein homologue Kes1p function in regulation of Sec14p-dependent protein transport from the yeast Golgi complex

2002 ◽  
Vol 157 (1) ◽  
pp. 63-78 ◽  
Author(s):  
Xinmin Li ◽  
Marcos P. Rivas ◽  
Min Fang ◽  
Jennifer Marchena ◽  
Bharat Mehrotra ◽  
...  
Keyword(s):  
Golgi Complex ◽  
Protein Transport ◽  
Adenosine Diphosphate ◽  
Functional Activities ◽  
Oxysterol Binding Protein ◽  
Golgi Region ◽  
Peripheral Membrane Protein ◽  
Dependent Protein ◽  
Altered Regulation

Oxysterol binding proteins (OSBPs) comprise a large conserved family of proteins in eukaryotes. Their ubiquity notwithstanding, the functional activities of these proteins remain unknown. Kes1p, one of seven members of the yeast OSBP family, negatively regulates Golgi complex secretory functions that are dependent on the action of the major yeast phosphatidylinositol/phosphatidylcholine Sec14p. We now demonstrate that Kes1p is a peripheral membrane protein of the yeast Golgi complex, that localization to the Golgi complex is required for Kes1p function in vivo, and that targeting of Kes1p to the Golgi complex requires binding to a phosphoinositide pool generated via the action of the Pik1p, but not the Stt4p, PtdIns 4-kinase. Localization of Kes1p to yeast Golgi region also requires function of a conserved motif found in all members of the OSBP family. Finally, we present evidence to suggest that Kes1p may regulate adenosine diphosphate-ribosylation factor (ARF) function in yeast, and that it may be through altered regulation of ARF that Kes1p interfaces with Sec14p in controlling Golgi region secretory function.

scholarly journals Phospholipid flippase activities and substrate specificities of human type IV P-type ATPases localized to the plasma membrane.

2016 ◽  
Vol 291 (41) ◽  
pp. 21421-21421 ◽  
Author(s):  
Hiroyuki Takatsu ◽  
Gaku Tanaka ◽  
Katsumori Segawa ◽  
Jun Suzuki ◽  
Shigekazu Nagata ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Substrate Specificities ◽  
Human Type ◽  
Type Iv ◽  
P Type ◽  
Phospholipid Flippase
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