Ift88, but not Kif3a, is required for establishment of the periciliary membrane compartment

ABSTRACT We have previously reported that Nef specifically interacts with a small but highly active subpopulation of p21-activated kinase 2 (PAK2). Here we show that this is due to a transient association of Nef with a PAK2 activation complex within a detergent-insoluble membrane compartment containing the lipid raft marker GM1. The low abundance of this Nef-associated kinase (NAK) complex was found to be due to an autoregulatory mechanism. Although activation of PAK2 was required for assembly of the NAK complex, catalytic activity of PAK2 also promoted dissociation of this complex. Testing different constitutively active PAK2 mutants indicated that the conformation associated with p21-mediated activation rather than kinase activity per se was required for PAK2 to become NAK. Although association with PAK2 is one of the most conserved properties of Nef, we found that the ability to stimulate PAK2 activity differed markedly among divergent Nef alleles, suggesting that PAK2 association and activation are distinct functions of Nef. However, mutations introduced into the p21-binding domain of PAK2 revealed that p21-GTPases are involved in both of these Nef functions and, in addition to promoting PAK2 activation, also help to physically stabilize the NAK complex.

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Relationship between lipid saturation and lipid-protein interaction in liver mitochondria modified by catalytic hydrogenation with reference to cardiolipin molecular species

Biochemical Journal ◽

10.1042/bj2650079 ◽

1990 ◽

Vol 265 (1) ◽

pp. 79-85 ◽

Cited By ~ 58

Author(s):

M Schlame ◽

L Horvàth ◽

L Vìgh

Keyword(s):

Double Bond ◽

Stearic Acid ◽

Liver Mitochondria ◽

Solvation Shell ◽

Fatty Acid Analysis ◽

Structural Requirement ◽

Hydrophobic Moiety ◽

Membrane Compartment ◽

Lipid Protein Interaction ◽

Tight Association

Lipid acyl double bonds in isolated mitochondrial membranes were gradually reduced by palladium-complex-catalysed hydrogenation, and the resulting saturation was monitored by fatty acid analysis of phosphatidylcholine, phosphatidylethanolamine and cardiolipin. The courses of hydrogenation of these phospholipids suggested that cardiolipin is in a membrane compartment which is less accessible to the applied catalyst. Native cardiolipin and its hydrogenation products were further characterized by analysis of their molecular diacylglycerol species. A decrease in the double bond content was accompanied by an increased amount of motionally restricted lipids at the hydrophobic interface of proteins as measured by two different spin-labelled lipids (C-14 positional isomers of spin-labelled stearic acid and phosphatidylcholine analogues). The protein-immobilized fraction of spin-labelled stearic acid increased in parallel with the hydrogenation of cardiolipin rather than of phosphatidylcholine or phosphatidylethanolamine. These data are interpreted in terms of a tight association of cardiolipin with membrane proteins, which becomes looser upon double bond reduction leading to the replacement of cardiolipin by spin-labelled stearic acid in the solvation shell. Thus the hydrophobic moiety of cardiolipin, characterized by double-unsaturated C18-C18 diacylglycerol species, seems to be an important structural requirement for the high protein affinity of this compound.

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Condensation of Bioactive Compounds into the Membrane Compartment by Conjugation with Synthetic Polypeptides

Biomimetic Polymers ◽

10.1007/978-1-4613-0657-3_11 ◽

1990 ◽

pp. 203-222

Author(s):

Yukio Imanishi ◽

Shunsaku Kimura

Keyword(s):

Bioactive Compounds ◽

Synthetic Polypeptides ◽

Membrane Compartment

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Localization of lipophosphonoglycan in membranes of Acanthamoeba by using specific antibodies

Molecular and Cellular Biology ◽

10.1128/mcb.1.4.358-369.1981 ◽

1981 ◽

Vol 1 (4) ◽

pp. 358-369

Author(s):

C F Bailey ◽

B Bowers

Keyword(s):

Protein A ◽

Contractile Vacuole ◽

Membrane Perturbation ◽

Cytoplasmic Vacuoles ◽

Cytoplasmic Surface ◽

Antibody Method ◽

Membrane Compartment ◽

Vacuolar Membranes ◽

Indirect Immunofluorescent ◽

Unlabeled Antibody

Antibodies against two electrophoretically distinct forms of lipophosphonoglycan (LPG) were produced in rabbits. Antibody specificity was demonstrated by the coupled antibody 125I-protein A assay (Adair et al., J. Cell Biol. 79:281-285, 1978). Indirect immunofluorescent labeling of intact Acanthamoeba showed that antibodies to both LPG components had the same uniform distribution on the cell surface. Both antibodies also bound to the cytoplasmic surface of isolated phagosomes. The location of LPG in other membranes of the amoeba was demonstrated on sections by the unlabeled antibody method. Although LPG was absent from the nuclear membrane, virtually all of the internal vacuole membranes were labeled, including the contractile vacuole. Antibodies directed against LPG were utilized to label lipophosphonoglycan in the plasma membrane of living amoebae. Labeled membrane was internalized and then localized by immunofluorescence in cytoplasmic vacuoles within 10 min of incubation. Although these results are evidence for exchange between plasma and cytoplasmic vacuolar membranes, the contractile vacuole remained unlabeled and can be considered, therefore, a separate membrane compartment. Concanavalin A also was bound and internalized by the amoeba, but electron microscopy showed that this label caused pronounced membrane perturbation, limiting its usefulness as a membrane marker in this system.

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Novel syntaxin gene sequences from Giardia, Trypanosoma and algae: implications for the ancient evolution of the eukaryotic endomembrane system

Journal of Cell Science ◽

10.1242/jcs.115.8.1635 ◽

2002 ◽

Vol 115 (8) ◽

pp. 1635-1642 ◽

Cited By ~ 3

Author(s):

Joel B. Dacks ◽

W. Ford Doolittle

Keyword(s):

Protein Interactions ◽

Calcium Binding ◽

Parallel Evolution ◽

Membrane System ◽

Compartment System ◽

Eukaryotic Diversity ◽

Wide Range ◽

Membrane Compartment ◽

Isoleucine Residue

SNAP receptors or SNARES are crucial components of the intracellular membrane system of eukaryotes. The syntaxin family of SNAREs have been shown to have roles in neurotransmission, vesicular transport, membrane fusion and even internal membrane compartment reconstruction. While syntaxins and SNAREs in general have been well characterized in mammalian and yeast models, little is known about their overall distribution across eukaryotic diversity or about the evolution of the syntaxin gene family. By combining bioinformatic,molecular biological and phylogenetic approaches, we demonstrate that various syntaxin homologs are not only present in `eukaryotic crown taxa' but across a wide range of eukaryotic lineages. The alignment of evolutionarily diverse syntaxin paralogs shows that an isoleucine residue critical to nSec1—syntaxin complex formation and the characteristic syntaxin glutamine residue are nearly universally conserved, implying a general functional importance for these residues. Other identified functional residues involved in botulism toxicity and calcium-binding-protein interactions are also compared. The presence of Golgi-related syntaxins in the intestinal parasite Giardia intestinalis provides further evidence for a cryptic Golgi in this `adictyosomal' taxon, and another likely case of secondary reduction in this parasite. The phylogeny of syntaxins shows a number of nested duplications, including a case of parallel evolution in the plasma membrane-associated syntaxins, and ancestral duplications in the other syntaxin paralogs. These speak to ancient events in the evolution of the syntaxin system and emphasize the universal role of the syntaxins in the eukaryotic intracellular compartment system.

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Division Plane Establishment and Cytokinesis

Annual Review of Plant Biology ◽

10.1146/annurev-arplant-050718-100444 ◽

2019 ◽

Vol 70 (1) ◽

pp. 239-267 ◽

Cited By ~ 17

Author(s):

Pantelis Livanos ◽

Sabine Müller

Keyword(s):

Secretory Pathway ◽

Preprophase Band ◽

Cell Plate ◽

Division Plane ◽

Spatial Reference ◽

Division Site ◽

Eukaryotic Proteins ◽

Plate Formation ◽

Membrane Compartment ◽

Cytoplasmic Content

Plant cells divide their cytoplasmic content by forming a new membrane compartment, the cell plate, via a rerouting of the secretory pathway toward the division plane aided by a dynamic cytoskeletal apparatus known as the phragmoplast. The phragmoplast expands centrifugally and directs the cell plate to the preselected division site at the plasma membrane to fuse with the parental wall. The division site is transiently decorated by the cytoskeletal preprophase band in preprophase and prophase, whereas a number of proteins discovered over the last decade reside continuously at the division site and provide a lasting spatial reference for phragmoplast guidance. Recent studies of membrane fusion at the cell plate have revealed the contribution of functionally conserved eukaryotic proteins to distinct stages of cell plate biogenesis and emphasize the coupling of cell plate formation with phragmoplast expansion. Together with novel findings concerning preprophase band function and the setup of the division site, cytokinesis and its spatial control remain an open-ended field with outstanding and challenging questions to resolve.

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A model for Rab GTPase localization

Biochemical Society Transactions ◽

10.1042/bst0330627 ◽

2005 ◽

Vol 33 (4) ◽

pp. 627-630 ◽

Cited By ~ 48

Author(s):

S. Pfeffer

Keyword(s):

Steady State ◽

Cellular Localization ◽

Rab Gtpase ◽

Rab Proteins ◽

Rab Gtpases ◽

Macromolecular Assemblies ◽

Complex Interactions ◽

Membrane Compartment ◽

Membrane Bound ◽

Distinct Membrane

The human genome encodes almost 70 Rab GTPases. These proteins are C-terminally geranylgeranylated and are localized to the surfaces of distinct membrane-bound compartments in eukaryotic cells. This mini review presents a working model for how Rabs achieve and maintain their steady-state localizations. Data from a number of laboratories suggest that Rabs participate in the generation of macromolecular assemblies that generate functional microdomains within a given membrane compartment. Our data suggest that these complex interactions are important for the cellular localization of Rab proteins at steady state.

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Haustorium Formation in Medicago truncatula Roots Infected by Phytophthora palmivora Does Not Involve the Common Endosymbiotic Program Shared by Arbuscular Mycorrhizal Fungi and Rhizobia

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-06-15-0130-r ◽

2015 ◽

Vol 28 (12) ◽

pp. 1271-1280 ◽

Cited By ~ 18

Author(s):

Rik Huisman ◽

Klaas Bouwmeester ◽

Marijke Brattinga ◽

Francine Govers ◽

Ton Bisseling ◽

...

Keyword(s):

Medicago Truncatula ◽

Mycorrhizal Fungi ◽

Arbuscular Mycorrhizal ◽

Am Fungi ◽

Phytophthora Palmivora ◽

Living Plant ◽

Mutualistic Symbiosis ◽

Membrane Compartment ◽

Microbe Interactions ◽

Host Genes

In biotrophic plant-microbe interactions, microbes infect living plant cells, in which they are hosted in a novel membrane compartment, the host-microbe interface. To create a host-microbe interface, arbuscular mycorrhizal (AM) fungi and rhizobia make use of the same endosymbiotic program. It is a long-standing hypothesis that pathogens make use of plant proteins that are dedicated to mutualistic symbiosis to infect plants and form haustoria. In this report, we developed a Phytophthora palmivora pathosystem to study haustorium formation in Medicago truncatula roots. We show that P. palmivora does not require host genes that are essential for symbiotic infection and host-microbe interface formation to infect Medicago roots and form haustoria. Based on these findings, we conclude that P. palmivora does not hijack the ancient intracellular accommodation program used by symbiotic microbes to form a biotrophic host-microbe interface.

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Chicken Erythroid AE1 Anion Exchangers Associate with the Cytoskeleton During Recycling to the Golgi

Molecular Biology of the Cell ◽

10.1091/mbc.10.2.455 ◽

1999 ◽

Vol 10 (2) ◽

pp. 455-469 ◽

Cited By ~ 17

Author(s):

Sourav Ghosh ◽

Kathleen H. Cox ◽

John V. Cox

Keyword(s):

Plasma Membrane ◽

Ammonium Chloride ◽

Anion Exchanger ◽

Secretory Pathway ◽

Brefeldin A ◽

Endocytic Pathway ◽

Erythroid Cells ◽

Anion Exchangers ◽

Membrane Compartment ◽

Chloride Treatment

Chicken erythroid AE1 anion exchangers receive endoglycosidase F (endo F)-sensitive sugar modifications in their initial transit through the secretory pathway. After delivery to the plasma membrane, anion exchangers are internalized and recycled to the Golgi where they acquire additional N-linked modifications that are resistant to endo F. During recycling, some of the anion exchangers become detergent insoluble. The acquisition of detergent insolubility correlates with the association of the anion exchanger with cytoskeletal ankyrin. Reagents that inhibit different steps in the endocytic pathway, including 0.4 M sucrose, ammonium chloride, and brefeldin A, block the acquisition of endo F-resistant sugars and the acquisition of detergent insolubility by newly synthesized anion exchangers. The inhibitory effects of ammonium chloride on anion exchanger processing are rapidly reversible. Furthermore, AE1 anion exchangers become detergent insoluble more rapidly than they acquire endo F-resistant modifications in cells recovering from an ammonium chloride block. This suggests that the cytoskeletal association of the recycling anion exchangers occurs after release from the compartment where they accumulate due to ammonium chloride treatment, and prior to their transit through the Golgi. The recycling pool of newly synthesized anion exchangers is reflected in the steady-state distribution of the polypeptide. In addition to plasma membrane staining, anion exchanger antibodies stain a perinuclear compartment in erythroid cells. This perinuclear AE1-containing compartment is also stained by ankyrin antibodies and partially overlaps the membrane compartment stained by NBD C6-ceramide, a Golgi marker. Detergent extraction of erythroid cells in situ has suggested that a substantial fraction of the perinuclear pool of AE1 is cytoskeletal associated. The demonstration that erythroid anion exchangers interact with elements of the cytoskeleton during recycling to the Golgi suggests the cytoskeleton may be involved in the post-Golgi trafficking of this membrane transporter.

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Differential Localization of Rho Gtpases in Live Cells

The Journal of Cell Biology ◽

10.1083/jcb.152.1.111 ◽

2001 ◽

Vol 152 (1) ◽

pp. 111-126 ◽

Cited By ~ 472

Author(s):

David Michaelson ◽

Joseph Silletti ◽

Gretchen Murphy ◽

Peter D'Eustachio ◽

Mark Rush ◽

...

Keyword(s):

Rho Gtpases ◽

Fluorescent Protein ◽

Essential Feature ◽

Hypervariable Region ◽

Dominant Negative ◽

Live Cells ◽

Rho Proteins ◽

Guanine Nucleotide Dissociation Inhibitor ◽

Membrane Compartment ◽

Green Fluorescent

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)α. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDIα in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDIα. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDIα binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDIα and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

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