Thesis
Reference
The intracellular transport of glycosylphosphatidylinositol-anchored proteins in mammalian Chinese Hamster Ovary cells
JAENSCH, Nina
Abstract
GPI-APs are a class of lipid-anchored proteins expressed on the cell surface of eukaryotes.
Their potential interaction with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in their intracellular transport. Here, we examined the importance of two saturated fatty acids within the phosphatidylinositol moiety of GPI-APs.
These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. The fatty acid remodeling is not required for both, efficient Golgi-to-plasma membrane transport and selective endocytosis via CLIC/GEEC pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence seems not related to the potential interaction with ordered lipid domain mediated by two saturated fatty acids.
JAENSCH, Nina. The intracellular transport of glycosylphosphatidylinositol-anchored proteins in mammalian Chinese Hamster Ovary cells . Thèse de doctorat : Univ. Genève, 2014, no. Sc. 4676
URN : urn:nbn:ch:unige-384155
DOI : 10.13097/archive-ouverte/unige:38415
Available at:
http://archive-ouverte.unige.ch/unige:38415
Disclaimer: layout of this document may differ from the published version.
1 / 1
UNIVERSITÉ DE GENÈVE FACULTÉ DES SCIENCES Section de chimie et biochimie
Département de biochimie Professeur Jean Gruenberg Professeure Reika Watanabe
The Intracellular Transport of Glycosylphosphatidylinositol-Anchored Proteins in Mammalian Chinese Hamster Ovary Cells
THÈSE
présentée à la faculté des sciences de l`Université de Genève pour obtenir le grade de Docteur ès sciences, mention biochimie
par Nina JAENSCH
de
Ratzeburg (Allemagne)
Thèse no. 4676
Genève
Ateliers d´impression - Repromail
2014
Acknowledgements
It was a long journey and I would like to thank everyone who was part of it and who helped me along the way.
I would like to express my appreciation and thanks to my advisor Dr. Reika Watanabe. I thank you for the opportunity to work on this project, for your advice and effort on my work and for passing on your passion for science. I am thankful for the time we spent together and for the experience I gained while working in your lab. I thank you very much for coming all the way from California for my thesis defense, it really meant a lot to me.
I also deeply thank Professor Dr. Jean Gruenberg for taking me over as a PhD student when Reika left. Your advice and encouragement on my thesis and my research as well as on on my career has been priceless.
I would also like to thank my committee members Professor Dr. Howard Riezman and Professor Dr. Chiara Zurzolo. I highly value your scientific opinion on my thesis and I am very grateful for your comments and suggestions.
I would like to express my gratitude to all the members of the biochemistry department, the bioimaging platform and the PhD program that provided a wonderful and motivating work environment. It has been a pleasure to work with all of you.
I would like to thank Daniel Abegg who always supported me with helpful discussions and advice.
Special thanks to my wonderful friends Britta Knight, Boris Lee and Colette Berry. Thank you for always believing in me, for your advice, encouragement, and your support in achieving my goals.
Ich danke meinen Eltern Kinschi und Steffi Jaensch, meiner Schwester und meinem Schwager
Tini und Karsten Becker. Ihr habt an mich geglaubt und ward immer an meiner Seite.
Abbreviations
AP-1 Adaptor protein-1 AP-2 Adaptor protein-2 AP-4 Adaptor protein-4 BAR Bin–Amphiphysin–Rvs BHK Baby Hamster Kidney CCP Clathrin-coated pits CCV Clathrin-coated vesicles CHO Chinese Hamster Ovary
CIE Clathrin-independent endocytosis CLIC Clathrin-independent carrier CME Clathrin-mediated endocytosis COP I Coat protein complex I
COP II Coat protein complex II CTxB Cholera toxin B
DAF Decay-acceleration factor DRM Detergent-resistant membrane
EE Early endosome
EEA-1 Early endosome antigen-1 ER Endoplasmic Reticulum ERES ER exit sites
EtNP Ethanolaminephosphate FR Folate receptor
FRET Fluorescent resonance energy transfer GAP GTPase activating protein
GEEC GPI-enriched endocytic compartment GFP Green fluorescent protein
GH Growth hormone
GlcN Glucosamine
GPI Glycosylphosphatidylinositol
GPI-APs Glycosylphosphatidylinositol-anchored proteins GUVs Giant unilammellar vesicles
HA Hemagglutinin
IL2-Rβ Interleukin-2 receptor
KD Knockdown
LDLR Low-density lipoprotein receptor
LE Late endosome
LFA-3 Lymphocyte-function-associated antigen-3
LL dileucine
l
dLiquid-disordered l
oLiquid-ordered
LRP low-density lipoprotein receptor-related proteins MßCD Methyl-β-cyclodextrin
MDCK Madin Darby canine kidney
MesNa Sodium 2-mercaptoethane-sulfonate MHC I Major histocompatibility protein class I
Man Mannose
M6P Mannose-6-phosphate
M6PR Mannose-6-phosphate receptor NRK Normal rat kidney
PE-PEG Phosphatidylethanolamine-polyethyleneglycol PH Pleckstrin homology
PGAP post-GPI-attachment to proteins PLA
2Phospholipase A
2PLAP Placental alkaline phosphatase PLD Phospholipase D
PI Phosphatidylinositol
PI4P phosphatidylinositol-4-phosphate PI4,5P
2Phosphatidylinositol-4,5-bisphosphate PIG Phosphatidylinositol glycan
PI-PLC Phosphatidylinositol-specific phospholipase
PM Plasma membrane
PNH Paroxysmal nocturnal hemoglobinuria PrP Prion protein
PrP
CCellular prion protein PrP
ScScrapie prion protein
PTRF Polymerase I and transcript release factor RE Recycling endosome
SF-CD59 SNAP-Flag-CD59
SM sphingomyelin
src sarcoma
SV40 Simian virus 40
TA Transamidase
Tf Transferrin
TfR Transferrin receptor TGN Trans-Golgi network
TIRF Total internal reflection fluorescence
TM Transmembrane
uPA Urokinase type plasminogen activator
uPAI Urokinase type plasminogen activator inhibitor
uPAR Urokinase type plasminogen activator receptor
VSVG Vesicular stomatitis virus glycoprotein
Table of contents
Abstract………...1
Résumé (francais)……….3
I Introduction ……….5
Glycosylphosphatidylinositol-anchored proteins………..5
Physiological functions of GPI-anchored proteins………6
GPI-anchor remodeling………..8
Association of GPI-APs with ordered lipid domains………9
The secretory pathway of GPI-anchored proteins………..12
ER-to-Golgi transport………...12
Golgi-to-plasma membrane transport………...14
Endocytosis of GPI-anchored proteins………...16
Recycling of GPI-anchored proteins………19
Project goal………..19
II Results I ………...21
Manuscript: Exit of GPI-anchored proteins from the ER differs in yeast and mammalian cells….23 III Results II ………...41
Manuscript: Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent in remodeled GPI………...43
IV Results III ………..59
Manuscript: Stable expression but not transport depends on the fatty acid structure of GPI- anchored proteins………...61
Appendix I: The SNAP-tag technology to study transport of GPI-anchored proteins……….101
Appendix II: The association of GPI-anchored proteins with ordered lipid domains………103
V Discussion ………...105
ER-to-Golgi transport of GPI-anchored proteins………...105
GPI-anchored protein sorting in yeast and mammalian cells………..106
Golgi-to-plasma membrane transport of GPI-anchored proteins………...107
The role of cholesterol in Golgi-to-plasma membrane transport………..109
Endocytosis of GPI-anchored proteins………110
Endocytosis of free GPI-anchored proteins……….110
Endocytosis of bound GPI-anchored proteins………...111
The role of cholesterol in endocytosis………113
Physiological role of lipid remodeling……….115
References ………...117
1
Abstract
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of lipid-anchored proteins that are expressed at the cell surface. In mammalian cells, more than 150 proteins are post- translationally modified by glycosylphosphatidylinositol (GPI) and have various important functions such as hydrolytic enzymes, receptors, adhesion molecules and immunologically relevant proteins for complement regulatory processes. Biogenesis of GPI is essential in
mammals and defects in GPI biosynthesis have been associated with various diseases [Nozaki et al., 1999; Ware et al., 1994; Almeida et al., 2006]. Efficient transport of GPI-APs to their final destinations is therefore critical to ensure proper functioning of these proteins. Sorting and concentration of cargo proteins during intracellular transport often depends on cytosolic motifs that are recognized by transport machineries. However, GPI-APs lack a cytosolic domain and are exoplasmically exposed, which makes them inaccessible for transport machineries located in the cytosol. This raises the exciting questions of how and if GPI-APs are concentrated for their efficient sorting into transport carriers and makes studies on the transport of GPI-APs both interesting and challenging. Sorting of GPI-APs is not very well understood, however, several different mechanisms have been suggested. Firstly, receptors that link GPI-APs to cytosolic transport machineries are thought to be involved in efficient packaging of GPI-APs into transport carriers [Bonnon et al., 2010]. Secondly, the GPI anchor has been proposed to function as sorting signal in the intracellular transport through a lipid-mediated mechanism that requires the interaction with ordered lipid domains enriched in cholesterol and sphingolipids [Simons and Ikonen, 1997]. Thirdly, oligomerization of GPI-APs is believed to facilitate their proper transport [Paladino et al., 2004; Suzuki et al., 2012]. An alternative hypothesis is a possible transport by bulk flow. In this study, I present and discuss new results on transport mechanisms of GPI-APs along the secretory and endocytic pathways in non-polarized Chinese Hamster Ovary (CHO) cells with a particular focus on the involvement of GPI anchor remodeling events which add new insight and knowledge to our current understanding. New results on the sorting of GPI-APs in the ER indicate that p24 family proteins act as cargo receptors exclusively for correctly remodeled GPI-APs for their sorting into coat protein complex II (COPII) vesicles. Two
remodeling reactions of the GPI anchor, mediated by post-GPI-attachment to proteins (PGAP) 1 and PGAP 5, are required for the recognition by p24 protein complexes and for sorting of GPI- APs into ER exit sites (ERES). We also found critical differences in the ER exit mechanism of GPI- APs between yeast and mammalian cells. Furthermore, our results on the Golgi exit and
endocytosis of GPI-APs provide evidence against a lipid-mediated sorting mechanism through
interaction with cholesterol and sphingolipid-enriched ordered lipid domains. GPI-APs associate
2 with ordered lipid domains, also known as lipid rafts, through saturated fatty acids within the phosphatidylinositol (PI) moiety of the GPI anchor [Simons and Ikonen, 1997]. The saturated fatty acids are introduced by the action of the lipid remodeling enzymes PGAP 2 and PGAP 3 in the Golgi apparatus and are believed to be essential for the association of GPI-APs with ordered lipid domains [Maeda et al., 2007; Fujita et al., 2006]. In this study, we address the potential role of lipid-mediated sorting of GPI-APs for their efficient Golgi exit and endocytosis by using mutant cells that are defective in lipid remodeling. Considering that lipid-mediated sorting of GPI-APs has been mainly tested by cholesterol and sphingolipid depletion, lipid remodeling mutants provide a new and valuable tool to challenge this hypothesis. We established two novel quantitative transport assays using the SNAP-tag labeling technology that allow us to measure the transport from the Golgi to the plasma membrane and endocytosis of GPI-APs. We found that the transport kinetics, as well as the sorting of GPI-APs into the GPI-enriched endocytic
compartments/ clathrin-independent carriers (GEEC/CLIC) pathway are identical for remodeled
and unremodeled GPI-APs and therefore independent of the association with ordered lipid
domains. We suggest that lipid remodeling and the resulting association of GPI-APs with ordered
lipid domains is not required for their efficient intracellular transport. In contrast, cholesterol
depletion affected the transport kinetics of GPI-APs in the secretory and endocytic pathway
which suggests that the mechanism of cholesterol dependence is not related to the sorting of
GPI-APs into ordered lipid domains. Furthermore, we identified an important physiological role
of the lipid remodeling. We found that cholesterol extraction drastically decreases the surface
expression of unremodeled GPI-APs, but not of remodeled GPI-APs which implies an essential
role of lipid remodeling in the stable association of GPI-APs at the cell surface, especially under
potential membrane lipid perturbation.
3
Résumé
Les protéines à ancrage glycosylphosphatidylinositol (GPI) sont une classe de protéines exprimées à la surface de la cellule et ancrées dans la membrane plasmique via un lipide. Dans les cellules de mammifères, plus de 150 protéines sont modifiées post-traductionnellement par l´ajout d´une ancre GPI. Ces protéines ont diverses activités comme, par exemple des enzymes hydrolytiques, des récepteurs, des molécules d´adhésion et des protéines immunologiques impliquées dans la régulation du système du complément. La biogenèse de l’ancre GPI est essentielle chez les mammifères et des défauts dans sa biosynthèse sont associées avec diverses maladies [Nozaki et al., 1999; Ware et al., 1994; Almeida et al., 2006]. De ce fait, un transport efficace des protéines à ancrage GPI vers leurs destinations finales est critique pour assurer leur bon fonctionnement. Tri et concentration de protéines cargo durant le transport intracellulaire dépendent souvent de motifs cytoplasmiques reconnus par les machineries de transport. A l´inverse, les protéines à ancrage GPI n’ont pas de motif cytoplasmique et sont physiquement dans le lumen des organelles ou exposées à l’extérieur de la cellule. De ce fait, les protéines à ancrage GPI sont inaccessibles aux machineries de transport présentes dans le cytoplasme. Cela soulève donc les questions de comment les protéines à ancrage GPI sont efficacement
incorporées, avec ou sans mécanisme de concentration, dans des vésicules de transport et de part ce fait rend l’étude du transport de ces protéines intéressant et stimulant. Le tri des protéines à ancrage GPI n’est pas totalement élucidé, mais plusieurs mécanismes ont été suggérés. Premièrement, des récepteurs liant les protéines à ancrage GPI aux machineries présentes dans le cytoplasme participent à une incorporation efficace dans les vésicules de transport [Bonnon et al., 2010]. Deuxièmement, l’ancre GPI elle-même fonctionnerait comme un signal de tri lipidique pour le transport intracellulaire grâce à son interaction avec des
microdomaines lipidique ordonnés riche en cholestérol et sphingolipides [Simons and Ikonen, 1997]. Troisièmement, l’oligomérisation des protéines à ancrage GPI est proposée pour faciliter leur transport [Paladino et al., 2004; Suzuki et al., 2012]. Une hypothèse alternative est un transport par « bulk flow ». Dans cette étude, je présente et discute de nouveaux résultats sur les mécanismes de transport des protéines à ancrage GPI dans la voie de sécrétion et de
l’endocytose dans des cellules non polarisé CHO (« Chinese Hamster Ovary »). Une attention
particulière est portée sur les différents évènements de remodelage de l’ancre GPI, amenant de
nouvelles perspectives et connaissances dans la compréhension du transport intercellulaire de
ces protéines. De récents résultats sur le tri des protéines à ancrage GPI dans le RE indiquent
que les protéines de la famille des p24 participent comme récepteurs exclusifs pour les
protéines dont l’ancre GPI a été correctement remodelée, pour leur incorporation dans les
4 vésicules « coat protein complexes II » (COPII) au niveau des sites de sortie du RE (ERES). Deux réactions de remodelage de l’ancre GPI sont nécessaires pour cette reconnaissance et impliquent les protéines « post-GPI-attachment to proteins » (PGAP) 1 et PGAP 5. Nous avons aussi trouvé d’importantes différences dans le mécanisme de sortie du RE des protéines à ancrage GPI entre la levure et les cellules de mammifères. Contrairement à la levure, la sortie du RE dans les cellules de mammifères des protéines à ancrage GPI est étroitement dépendante de la GTPase Sar1 et indépendante de la synthèse de sphingolipides. En outre, nos résultats sur la sortie de l’appareil de Golgi et de l’endocytose des protéines à ancrage GPI présentent des preuves
infirmant un tri lipidique lié à l’interaction avec des microdomaines lipidiques ordonnés riche en cholestérol et sphingolipides. Les protéines à ancrage GPI sont incorporées dans des
microdomaines lipidiques ordonnée, les « lipid raft », grâce aux acides gras saturés présents dans le phosphatidylinositol (PI) de l’ancre GPI [Simons and Ikonen, 1997]. Le remodelage lipidique de l’ancre GPI dans l’appareil de Golgi comporte le remplacement d’un acide gras insaturé par un acide gras saturé par les enzymes PGAP 2 et PGAP 3. Ce remodelage est supposé essentiel pour l’incorporation des protéines à ancrage GPI dans les microdomaines lipidiques ordonnés [Maeda et al., 2007; Fujita et al., 2006]. Dans cette étude, nous examinons le rôle potentiel du tri lipidique des protéines à ancrage GPI pour leur sortie de l’appareil de Golgi ainsi que pour leur endocytose, à l’aide de mutants déficients dans le remodelage lipidique. Ces mutants sont un nouvel outil pour réévaluer l’hypothèse du tri lipidique, qui jusqu’à présent était étudiée par la déplétion du cholestérol et des sphingolipides. Nous avons établis deux nouvelles techniques quantitatives utilisant le marquage « SNAP » pour suivre le transport des protéines à ancrage GPI entre l’appareil de Golgi et la membrane plasmique ainsi que dans la voie d’endocytose. Nous avons trouvé que les cinétiques de transport ainsi que le tri dans les
« GPI-enriched endocytic compartments/ clathrin-independent carriers » (GEEC/CLIC) sont identiques pour les protéines dont l’ancre GPI est ou n’est pas remodelé. Nous suggérons donc la non-nécessité du remodelage lipidique de l’ancre GPI, ainsi que l’incorporation résultante avec les microdomaines lipidiques ordonnés pour un transport intracellulaire efficace des protéines à ancrage GPI. En revanche, la réduction de cholestérol affecte les cinétiques de transport dans les voies de sécrétion et d’endocytose, indiquant que la dépendance au cholestérol n’est pas liée à l’incorporation des protéines à ancrage GPI dans des microdomaines lipidiques ordonnés. Nous avons aussi identifié un important rôle physiologique du remodelage lipidique de l’ancre GPI.
Nous avons trouvé que l’extraction de cholestérol diminue drastiquement l’expression à la
membrane plasmique des protéines dont l’ancre GPI n’a pas subi le remodelage lipidique,
indiquant un rôle essentiel de celui-ci pour une incorporation stable de ces protéines dans la
membrane plasmique, particulièrement lors de perturbation lipidique.
5
I Introduction
Glycosylphosphatidylinositol-anchored proteins
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a very diverse class of lipid- anchored proteins expressed at the cell surface of all eukaryotic cells from vertebrates to protozoa. In mammalian cells, more than 150 proteins are post-translationally modified by glycosylphosphatidylinositol (GPI) and anchored to the outer leaflet of the plasma membrane via their fatty acid moiety. GPI-APs have various functions, such as hydrolytic enzymes, receptors, adhesion molecules and immunologically relevant proteins such as complement regulatory proteins. GPI-APs were discovered in the late 1970s by the observation that alkaline phosphatase and acetylcholinesterase were released from cell membranes by a
phosphatidylinositol (PI)-specific phospholipase C (PI-PLC) [Low and Finean, 1977 and 1978].
The structure of GPI, however, was only characterized in 1985 [Futerman et al., 1985; Roberts and Rosenberry, 1985; Tse et al. 1985; Ferguson et al., 1988]. The conserved core structure of GPI consists of a PI, a glucosamine (GlcN) linked to the inositol, a linear chain of three mannose (Man) sugars linked to the glucosamine, and an ethanolaminephosphate (EtNP) linked to the terminal mannose (EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6PI) (Fig.1A).
The protein is attached to the GPI via an amide bond between the C-terminal residue of the protein and the amino group of EtNP. Aside from the core, the structure of the GPI anchor varies between species and cell types. GPI can be modified by the addition of side-branching groups to its glycan core and by remodeling of its lipid moiety. During biosynthesis, the GPI core is
modified by the addition of a palmitoyl group to the 2-OH of the inositol moiety and up to three
EtNPs to specific positions on the three mannoses (Fig.1B). The GPI anchor is synthesized in the
endoplasmic reticulum (ER) from PI by a stepwise transfer of sugars, a fatty acid and EtNP to
form a complete GPI precursor. The GPI biosynthesis is initiated on the cytoplasmic face of the
ER and completed in the ER lumen requiring transbilayer flipping of a glycolipid intermediate
[Vishwakarma et al., 2005] (Fig.1B).
6
Fig.1: Structure and biosynthetic pathway of mammalian GPI-anchored proteins. A) GPI-APs have a common core backbone: protein-EtNP-6Manα1-2Manα1-6Manα1-4GlcNα1-6PI. Various side-branches are linked to the mannose residues. GPI-APs from nucleated cells have two saturated acyl chains in their PI moiety, whereas those from human erythrocytes have an unsaturated acyl chain in the sn-2 position and an inositol-linked acyl chain. B) The biosynthesis of GPI is performed in the ER through an enzymatic reaction pathway consisting of 10 steps [Kinoshita et al., 2008]. Preformed GPI is attached to proteins by GPI transamidase (TA).
(Picture taken from Kinoshita et al., 2008)
Physiological functions of GPI-anchored proteins
The importance of GPI-APs in mammals is demonstrated by the fact that abrogation of GPI biogenesis results in embryonic lethality [Nozaki et al., 1999]. However, for the growth of mammalian cells in culture, GPI biosynthesis is dispensable. The biosynthesis requires the expression of more than 20 proteins such as the phosphatidylinositol glycan (PIG) proteins [Orlean et al., 2007]. Defects in different enzymes involved in GPI biosynthesis such as PIG-A and PIG-M cause paroxysmal nocturnal hemoglobinuria (PNH) and autosomal recessive GPI-anchor deficiency, respectively [Ware et al., 1994; Almeida et al., 2006]. PNH is characterized by deficient cell surface expression of several GPI-APs such as decay-acceleration factor (DAF or also called CD55) and CD59, which interfere with the complement system and in their absence patients display complement-mediated hemolysis. Complement in blood is constitutively
activated at a low level and can be rapidly amplified to provide an immunoglobulin-independent
pathway of activation. Cells are protected from the activated complement by GPI-APs such as
7 CD59 and DAF. Complement regulatory proteins act by binding directly to membrane-associated components of the complement cascade and thereby prevent further recruitment of components that induce lysis.
Similarly, autosomal recessive GPI-anchor deficiency decreases the surface expression of CD59 and other GPI-APs in hematopoetic cells which leads to venous thrombosis and seizures.
Mutations in PIG-V and PIG-N have also been reported to cause inherited GPI deficiencies [Krawitz et al., 2010; Maydan et al., 2011].
GPI-APs are involved in a wide range of important physiological processes such as nutrient uptake, the already mentioned complement regulatory reactions, virus entry, toxin receptors (pore-forming toxins aerolysin from Aeromonas hydrophila and alpha toxin from Clostridium septicum), neurogenesis, cell migration, wound healing and signaling [Ikezawa, 2002, Chan et al., 2001; Diep et al., 1998; Gordon et al., 1999; Ross et al., 1994; Andreasen et a., 2000; Fukushima et al., 2005; Abrami et al., 1998; Nelson et al. 1997].
The classical example of GPI-APs is the cell surface receptor folate receptor (FR), which exists in three different isoforms and the GPI-anchored α- and β-isoforms are expressed in placental tissue and the buccal carcinoma cell line (KB cells) and is responsible for the uptake of the vitamin folate [Ross et al., 1994]. The γ-isoform, in contrast, lacks the GPI-attachment signal and is secreted (Reddy et al., 1999). Folate is a co-factor for DNA replication enzymes and a substrate for thymidine synthesis and its uptake is therefore essential for the survival of all dividing cells.
FR has also been hijacked as virus receptor by filoviruses such as Ebola and Marburg viruses [Chan et al., 2001]. Similarly, several other GPI-APs also serve as virus receptors such as DAF, which was identified as co-receptor for coxsackieviruses B3 and A21 and for Enterovirus 70 and echovirus 7 [Shafren et al., 1997; Karnauchow et al., 1996; Bergelson et al., 1994].
Another well-known GPI-AP, the cellular prion protein (PrP
C), plays a dual role in the biology of the brain. On the one hand, it is involved in numerous physiological functions such as
neurogenesis and myelin maintenance [Steele et al., 2006; Bremer et al., 2010]. On the other
hand, it causes neurodegenerative spongiform encephalopathies through its conversion into the
scrapie isoform PrP
Scwhich results in the accumulation of amyloid plaques on neurons [Prusiner
and Kingsbury, 1985].
8
Fig.2: Remodeling of the GPI anchor in mammalian cells. The acyl chain linked to inositol is eliminated by PGAP1.
A side-chain EtNP attached to the second mannose is removed by PGAP5. After arrival at the Golgi, an unsaturated fatty acid at the sn-2 position in the PI moiety is removed by PGAP3 and replaced with a saturated fatty acid by PGAP2.
(Picture taken from Fujita et al., 2011)
GPI-anchor remodeling
The structure of GPI anchors before their attachment to proteins is different from the mature GPI-APs on the cell surface due to several remodeling events in the ER and the Golgi apparatus [reviewed in Fujita et al., 2012]. In an early step of GPI biosynthesis, an acyl-chain is transferred to the inositol ring of the GlcN-PI by mammalian PIG-W generating GlcN-(acyl)PI, which is required for the efficient completion of later steps in GPI biogenesis [Murakami et al., 2003]
(Fig.1B). However, this inositol-linked acyl-chain is only a transient component of GPI during biosynthesis and is removed soon after the attachment of GPI to proteins. This reaction is carried out by mammalian post-GPI-attachment to proteins (PGAP) 1 (yeast homolog Bst1p), a multi-spanning protein localized in the ER [Tanaka et al., 2004] (Fig.2). Knockout (KO) of Pgap1 in mice results in severe defects in development and fertilization [Ueda et al., 2007]. Most
Pgap1-KO mice die soon after birth and often display otocephaly, a developmental defect causing severely disturbed face and jaw shaping. Surviving male Pgap1-KO mice are infertile due
to the inability of the sperm to ascend to the oviduct and to attach to the egg. Furthermore, in the biosynthetic pathway of GPI, EtNP is added to the three mannose residues mediated by
mammalian PIG-N, PIG-G/PIG-F and PIG-O/PIG-F. The EtNP side-chain added to the second mannose is eliminated by PGAP 5 (yeast homologs CDC1p and TED1p) after GPI
attachment to proteins in the ER [Fujita et al., 2009] (Fig.2). PGAP5 contains a metal-dependent phosphoesterase motif in the luminal side of the membrane and requires manganese for its enzymatic activity.
In addition to the glycan moiety, the lipid part also undergoes remodeling. The fatty acid
remodeling of GPI takes place in the Golgi in mammalian cells and in the ER in yeast. An
9 unsaturated fatty acid (C18:1; C20:4 or C22:4 in mammalian cells) at the sn-2 position of the lipid is removed and replaced by a saturated fatty acid (C18:0 in mammalian cells) mediated by PGAP 3 (yeast homolog Per1p) and PGAP 2 (yeast homolog Gup1p) [Fujita et al., 2006; Maeda et al., 2007; Tashima et al., 2006; Bosson et al., 2006] (Fig.2). PGAP 3, which is involved in the generation of lyso-GPI-APs, does not have a lipase-like motif, but has several serine and histidine residues that are conserved in active sites of the phospholipase A
2(PLA
2) family. PGAP 3 is required for the lipase activity, but it is unclear whether it has enzymatic activity itself or whether it plays a regulatory role. Immunofluorescence microscopy of HA-tagged PGAP 3 expressed in Chinese Hamster Ovary (CHO) cells revealed that its main localization is in the Golgi with only a weak staining in the ER [Maeda et al., 2007]. Pgap3-KO mice show complex phenotypes such as male specific low birth rate, morphological abnormalities, abnormal reflexes and growth retardation [Murakami et al. 2012]. PGAP 2 is involved in the addition of the
saturated fatty acid to the sn-2 position of GPI, however, PGAP 2 might be a regulatory factor rather than the acyltransferase itself [Tashima et al., 2006]. Immunofluorescence microscopy analysis with overexpressed PGAP 2 in Normal Rat Kidney (NRK) cells showed that PGAP 2 is found mainly in the Golgi and to a lesser extent in the ER [Tashima et al., 2006]. Based on the localization of PGAP 3 and PGAP 2, the remodeling process is thought to occur in the Golgi apparatus in mammalian cells. In PGAP 2-deficient CHO cells, lyso-GPI-APs are transported to the cell surface and are sensitive to phospholipase D (PLD) and are rapidly secreted into the culture medium resulting in a drastic decrease surface expression [Tashima et al., 2006]. The physiological role of GPI-AP lipid remodeling is not fully understood. However, it seems reasonable to believe that lipid remodeling is required for the association of GPI-APs with ordered lipid domains, often called lipid rafts, which are putative membrane domains enriched in cholesterol and sphingolipids [Maeda et al., 2007; Fujita et al., 2006].
Association of GPI-anchored proteins with ordered lipid domains
Lipid rafts were originally introduced as a model to explain sphingolipid- and cholesterol-
enrichment in the apical membrane of polarized epithelial cells, which exhibit differences in
lipid and protein composition when compared to basolateral membranes [Simons and van Meer,
1988]. Most cell types generated by multicellular organisms are polarized. A characteristic
feature of polarized cells is the separation of their surface into distinct apical and basolateral
membranes which provide a protective barrier against the external environment and an
exchange interface. This is particularly well-described in epithelial and endothelial cells and in
neurons. The basolateral membrane connects adjacent cells and the underlying tissue, whereas
10
Fig.3: Model for the organization of ordered lipid domains in membranes.
Ordered lipid domains (red) segregate from other parts of the membrane (blue). A) Ordered lipid domains contain proteins attached to the exoplasmic leaflet of the bilayer by their GPI anchor, proteins binding to the cytoplasmic leaflet by acyl tails (such as the src-family kinase yes), or proteins associating through their
transmembrane domains, like the influenza virus protein hemagglutinin (HA). B) The lipid bilayer in ordered lipid domains is asymmetric with sphingolipids (red) enriched in the exoplasmic leaflet and glycerolipids (green) in the cytoplasmic leaflet.
Cholesterol (grey) is present in both leaflets and fills the space under the head groups of sphingolipids [Simons and Ikonen, 1997] (Picture taken from Simons and Ikonen, 1997)
the apical membrane faces the lumen of an internal organ. The two membranes are separated by tight junctions, which help to prevent mixing of apical and basolateral membrane components.
Sphingolipids and GPI-APs exhibit a characteristically polarized distribution, being preferentially targeted to the apical surface in polarized epithelial cells [Simons and van Meer, 1988; Brown and Rose, 1992]. In order to explain their preferential delivery to the apical domain, glyco- sphingolipids and cholesterol were proposed to cluster together and form tightly packed
membrane domains at the exoplasmic leaflet of the Golgi apparatus (Fig.3). These domains were
considered as sorting platforms that can selectively include proteins, such as GPI-APs, destined
for the apical surface [Simons and van Meer, 1988]. The hypothesis of lipid-based sorting
provided a novel concept highlighting lipids as active factors for membrane functionality as
opposed to proteins and protein-protein interactions. The hypothesis was later generalized as a
principle of membrane subcompartmentalization, not only involved in post-Golgi trafficking, but
also in endocytosis, several signaling pathways, and other membrane functions [Simons and
Ikonen, 1997; Simons and Toomre, 2000]. Cholesterol has the property to order the lipid bilayer
and to reduce the permeability by causing the acyl chains to become closely packed and the
bilayer to thicken. The lipid acyl chains cannot simply deform to allow movement of small
molecules across the bilayer, but the lipids can still move freely past each other. High cholesterol
containing membrane domains are thus termed liquid-ordered (l
o) and the low cholesterol
containing membrane domains liquid-disordered (l
d). Cholesterol preferentially interacts with
sphingolipids rather than with unsaturated phospholipids [Ramstedt and Slotte, 2002]. And
indeed, cholesterol and sphingolipids are present at high levels at the plasma membrane with
sphingolipids restricted to the outer leaflet [van Meer, 1989; Bretscher, 1973]. Based on model
11 bilayers that have the capacity to form l
oand l
ddomains, the lipid raft model proposes that cholesterol and sphingolipid of the outer leaflet of the plasma membrane are not evenly distributed, but form l
odomains that float in a l
dbilayer [Rietveld and Simons, 1998]. However, results from model membranes might not allow a precise prediction of the situation in living cells. Physiological bilayers in living cells contain lipids with a large heterogeneity in length and saturation of their acyl chains and contain a number of diverse integral membrane proteins which are difficult to be mimicked by simplified model membranes [Munro, 2003].
Apart from GPI-APs, double acylated proteins such as sarcoma (src)-family kinases, some transmembrane (TM) proteins such as the Influenza virus protein Hemagglutinin (HA) have been suggested to be associated with lipid rafts. With the development of new technology, the concept of lipid rafts has evolved significantly. Lipid rafts have also been called membrane nanoclusters, microdomains or ordered lipid domains and are presently defined as dynamic nanoscale cholesterol- and sphingolipid-enriched, ordered assemblies of specific proteins. The most recent model from Simons and Gerl proposes that nanoscale assemblies of cholesterol, sphingolipids and selective proteins such as GPI-APs are dynamic and fluctuate in composition and can coalesce into platforms through lipid-lipid, lipid-protein and protein-protein
oligomerization interactions in response to external signals or the initiation of membrane trafficking events [Simons and Gerl, 2010].
Ordered lipid domains have often been operationally defined by their detergent insolubilization in the cold in vitro and in particular by their resistance to Triton X-100 at 4°C and are therefore also referred to as detergent-resistant membranes (DRMs). Because of their high lipid content, DRMs float to a low density during gradient centrifugation, which has been used to identify proteins associated with ordered lipid domains [Brown and Rose, 1992]. However, it has been questioned whether DRMs truly reflect specific membrane domains existing in living cells [Munro, 2003]. Schuck et al. demonstrated that detergents differ considerately in their ability to selectively solubilize membrane proteins and to enrich sphingolipids and cholesterol, which makes it unlikely that different detergents reflect the exact same aspects of membrane
organization [Schuck et al., 2003]. Isolation of DRMs is nonetheless still believed to be a valuable tool for membrane analysis [Schuck et al., 2003].
Possible involvements of lipid-mediated sorting for GPI-APs often have been tested by
cholesterol depletion. Cholesterol is typically depleted by cholesterol biosynthesis inhibitors
and/ or by direct extraction with cyclodextrins. Although both may affect other lipids such as
phosphatidylinositol-4-phosphate (PI4P) at the Golgi or phosphatidylinositol-4,5-bisphosphate
(PI4,5P
2) at the plasma membrane, it is possible to demonstrate specificity by replenishing
12 cholesterol in depleted cells [Kwik et al., 2003; Lu et al., 2012]. Sphingolipid and cholesterol depletion cause mistargeting of GPI-APs in polarized epithelial cells which leads to their transport to both apical and basolateral membranes [Mays et al., 1995; Paladino et al., 2004].
Similarly, sphingolipid and cholesterol depletion affects the internalization and sorting of GPI- APs along the endocytic pathway demonstrating the requirement for these lipids [Chang et al., 1992; Stevens and Tang, 1997]. However, the exact mechanism of how these lipids function in the intracellular transport of GPI-APs remains elusive.
The model of lipid-mediated protein sorting is not only limited to intracellular sorting of GPI- APs. The simian virus 40 (SV40) and cholera toxin (CTx) both bind to the glycosphingolipid GM1 on the cell surface. The length and saturation of fatty acids in GM1 or its analogs influence the internalization of SV40 and the traffic of CTx from the plasma membrane to the trans-Golgi network (TGN) and the ER [Ewers et al., 2010; Chinnapen et al., 2012]. This demonstrates that the fatty acid composition of cellular GM1 affects the infectivity and toxicity of these pathogens.
The secretory pathway of GPI-anchored proteins
The secretory pathway provides the exit route that mediates transport and sorting of newly synthesized secretory cargo proteins from the ER, the site of their biosynthesis, through the Golgi apparatus to their final destinations by budding and fusion of transport vesicles [Palade, 1975; Rothman et al., 1996; Shekman et al., 1996]. The eukaryotic secretory pathway is responsible for the delivery of a large variety of proteins, around one third of all translated proteins, to their proper cellular destinations and is essential for cellular function and
multicellular development [Dancourt et al., 2010]. Malfunctioning of the secretory pathway is associated with a continuously increasing number of diseases and disorders with extremely diverse phenotypes [reviewed in Aridor et al., 2000 and Aridor et al., 2002]. Secretory cargo includes ER- or Golgi-resident proteins, membrane proteins, plasma membrane-destined and secretory proteins and usually requires a signal sequence [Jackson and Blobel, 1977]. Many proteins that are transported along the secretory pathway contain specific sorting signals for proper targeting.
ER-to-Golgi transport
Correctly folded and assembled cargo is packaged into coat protein complex II (COP II) vesicles
at ER exit sites (ERES) for transport to pre-Golgi or Golgi compartments. Coat protein complex I
(COP I) vesicles mediate retrograde vesicular transport from the Golgi to the ER to recycle
vesicle components. COP II is composed of the small GTPase Sar1, the Sec23-Sec24 complex and
the Sec13-Sec31 complex. Selective ER export is mediated by cytosolic transport motifs that
13 interact with Sec24. Similar to other secretory cargo such as TM proteins, GPI-APs are
transported from the ER, through the Golgi apparatus to the cell surface (Fig.4). However, unlike TM proteins, GPI-APs lack a cytosolic domain and are luminally exposed and therefore cannot interact directly with Sec24 or other components of the cytosolic transport machinery.
Consequently, this raised the question of how GPI-APs exit the ER, whether it is a passive transport by bulk flow or an active transport mediated by ER exit receptors. It was shown that the ER exit of GPI-APs is COP II-dependent [Rivier et al., 2010; Bonnon et al., 2010]. Mammalian cells express the four Sec24 isoforms Sec24A, Sec24B, Sec24C and Sec24D and GPI-APs were shown to have a preference for the Sec24C and Sec24D isoforms [Bonnon et al., 2010]. The link between Sec24 and GPI-APs is mediated by p24 proteins, a family of ~24 kDa type-I TM
proteins. Members of this protein family play an important role in the antero- and retrograde transport between the ER and the Golgi apparatus. In mammalian cells, the most studied family members are p23, p24, p25, p26 and p28 (several nomenclatures have been introduced and are used in parallel) [reviewed in Strating et al., 2009]. All p24 proteins share a similar domain architecture including a short cytoplasmic tail with several highly conserved motifs for binding to COP I and COP II [Stamnes et al., 1995; Dominguez et al., 1998; Béthune et al., 2006]. The p24- p23 protein complex exhibited the same preference as GPI-APs for the Sec24C and Sec24D isoforms for the ER export. Furthermore, immunoprecipitation experiments confirmed physical interaction between the GPI-APs CD59 and FR with the p24-p23 protein complex which suggests that the p24-p23 complex acts as a cargo receptor for GPI-APs by facilitating their export from the ER in a Sec24C- and Sec24D-selective manner [Bonnon et al., 2010].
The ER exit of GPI-APs in mammalian cells differs significantly from yeast [Rivier et al., 2010]. In yeast, GPI-APs are preferentially transported from the ER to the Golgi in COP II vesicle
populations that are distinct from COP II vesicles containing other secretory cargo such as
transmembrane proteins [Muñiz et al., 2001]. Consistently, GPI-APs were sorted into distinct
ERES populations prior to their exit from the ER [Castillon et al., 2009]. The sorting of GPI-APs
into ERES depends on Emp24p, the yeast homolog of mammalian p24 and proper GPI-anchor
remodeling by Bst1p and Per1p. Yeast strains deficient in GPI fatty acid remodeling are defective
in concentrating GPI-APs into ERES, resulting in delayed transport from the ER to the Golgi
[Castillon et al., 2009]. In contrast, the transport from the ER is not affected in mammalian
PGAP3-deficient cells [Maeda et al., 2007].
14
Fig.4: The secretory and endocytic pathway of GPI-anchored proteins. GPI-APs are transported from the ER to the Golgi in a COPII- and p24-dependent manner. GPI-APs are further transported from the Golgi to the plasma membrane. In various cell lines, GPI-APs are primarily internalized through the CLIC/GEEC pathway, an endocytic pathway that is regulated by actin and the small GTPases arf1 and cdc42. CLIC/GEEC structures have a significant tubular ring-shape. After internalization, membrane and proteins can be quickly recycled back for plasma membrane maintenance and homeostasis. CLIC/GEEC structures fuse with Rab5 early endosomes and GPI-APs can either be targeted to recycling endosomes or late endosomes.
Golgi-to-plasma membrane transport
The Golgi apparatus is a specialized intracellular organelle for protein processing and sorting and various posttranslational modifications such as glycosylation take place [Rothman, 1994].
Cargo molecules are sorted at the TGN into distinct pleiomorphic carriers that are targeted to different final destinations. The TGN is a complex tubular membrane network that emerges from the stacked Golgi cisternae. One principle of cargo sorting into transport carriers depends on recognition of specific sorting motifs in the cytosolic tail of transmembrane proteins by cytosolic adaptors and coat proteins [Bonifacino and Traub, 2003; Rodriguez-Boulan and Müsch, 2005]. In polarized cells, membrane proteins destined for delivery to the basolateral domains contain sorting signals in their cytosolic tail, such as YXXϕ (X, any amino acid; ϕ, hydrophobic amino acid) and LL (dileucine), which are recognized by adaptor protein-1 (AP-1) and adaptor protein- 4 (AP-4) at the TGN [Fölsch et al., 1999; Simmen et al., 2002]. Enzymes targeted to the
endosomal/ lysosomal system contain Mannose-6-phosphate (M6P) groups on their N-glycans
that act as sorting signals and are recognized by Mannose-6-phosphate receptors (M6PR)
located at the TGN [Braulke and Bonifacino, 2009]. In contrast, sorting signals for apically
15 destined proteins are less well characterized, however, N- and O-glycans may act as sorting signals [Alfalah et al., 1999; Rodriguez-Boulan and Gonzalez, 1999; Scheifferle et al., 1995]. GPI- APs are one of the first postulated apical sorting signals, but the underlying mechanism is unclear.
The post-Golgi transport of GPI-APs is not very well understood, but is studied primarily in polarized cells where most GPI-APs are selectively transported to the apical membrane [Brown and Rose, 1992; Paladino et al., 2006]. Non-polarized cells seem to have apical and basolateral cognate routes from the TGN to the plasma membrane [Yoshimori et al., 1996]. As introduced earlier, the association with ordered lipid domains has also been proposed to mediate the apical targeting of apically-destined proteins [Simons and Ikonen, 1997].
Certain exceptions to a selective apical delivery of GPI-APs were found such as non-preferential apical and basolateral delivery and preferential basolateral delivery of several GPI-APs in Fisher rat kidney cells [Zurzolo et al., 1993; Sarnataro et al., 2002]. It was therefore suggested that the association with ordered lipid domains is not sufficient for apical sorting [Paladino et al., 2004].
In addition to the association with ordered lipid domains, oligomerization of GPI-APs seems to be required for sorting into apical vesicles. It has been shown that in Madin Darby canine kidney (MDCK) cells both apically and basolaterally sorted GPI-APs are associated with DRMs which suggested their association with ordered lipid domains. GFP-GPI (GFP fused to the GPI
attachment signal of FR) and placental alkaline phosphatase (PLAP) were used as apically sorted model cargo proteins and GH-GPI (rat growth hormone fused to the GPI attachment signal of DAF) and the native PrP were used as basolaterally sorted model cargo proteins [Paladino et al., 2004]. This indicates that the association with ordered lipid domains might not provide an exclusive mechanism that mediates apical sorting. However, it has been shown that only apically sorted GPI-APs are able to oligomerize into high molecular weight complexes. Impairment of oligomerization leads to protein missorting to the basolateral domains [Paladino et al., 2004].
In CV-1 (normal African green monkey cells) cells and in HeLa cells, it has been proposed that GPI-APs and caveolin-1 are transported from the Golgi to the plasma membrane in the same transport carriers [Hayer et al., 2010]. Coexpressed caveolin-1 and GFP-GPI accumulate in the Golgi after a 20°C block and colocalize with the medial Golgi marker giantin, but not with
vesicular stomatitis virus glycoprotein (VSVG)-GFP or the TGN marker TGN46. It is possible that caveolin-1 and GFP-GPI carriers form in the medial Golgi and not in the TGN as other carriers.
The export step, measured after a temperature shift from 20°C to 37°C, was shown to be dependent on PI4P. Caveolin-1 and GFP-GPI containing vesicles en route to the plasma
membrane differ from those carrying VSVG-GFP in being uniformly small and less likely to have
16 a tubular shape. Upon arrival at the plasma membrane, measured by total internal reflection fluoresecence (TIRF) microscopy, GFP-GPI diffuse rapidly away, whereas caveolin-1 remain in place [Hayer et al., 2010].
Endocytosis of GPI-anchored proteins
Endocytosis describes the internalization of lipids, proteins and extracellular fluid into the cell through the production of internal membranes from the plasma membrane. The interplay between endocytosis and secretion allows a regulated interaction between the cell and its environment and the control of the lipid and protein composition of the plasma membrane.
Endocytosis also plays a regulatory role in cellular processes such as mitosis, antigen
presentation, cell migration and signaling [Doherty and McMahon, 2009; Hoeller et al., 2005].
Furthermore, endocytosis pathways are exploited by many viruses and other pathogens to mediate their entry into the cell [Marsh and Helenius, 2006]. Several endocytic pathways have been described, but some are still very poorly characterized. Clathrin-mediated endocytosis (CME) is the best characterized endocytic process and functions in eukaryotes for the selective internalization of cell surface molecules and extracellular material [Doherty and McMahon, 2009]. It is a highly coordinated process that begins with the assembly of clathrin coat
components through interactions with PI4,5P
2, adaptor proteins such as the adapter protein-2 (AP-2) and cargo proteins destined for internalization by this pathway. The nascent bud grows and invaginates with the assistance of accessory factors until the vesicle is released into the cytoplasm through a fission reaction mediated by the GTPase dynamin [Ferguson and De Camilli, 2012]. Cargo proteins internalized by CME carry binding motifs on their cytoplasmic tails which are recognized by adaptor proteins [Sorkin, 2004; Marks et al., 1996]. Distinct adaptors and accessory proteins control the internalization of distinct cargos which results in the formation of vesicle subtypes [reviewed in Doherty and McMahon, 2009]. Other less well studied endocytic pathways function independent of clathrin such as cavaolae, clathrin-independent carrier/ GPI- enriched endocytic compartment (CLIC/GEEC), arf6-dependent, flotillin-dependent or
macropinocytosis pathways. It is not clear whether all endocytic pathways exist in a constitutive manner. The classification of endocytic pathways is based on requirements for certain lipids and proteins, differential drug sensitivities, ultrastructural morphology and abilities to select
particular cargos. In contrast to the regular shaped clathrin-coated vesicles (CCV) with a size of
around 100 nm, clathrin-independent endocytosis (CIE) displays a diverse array of carrier
morphologies.
17 Early studies showed that GPI-APs might be internalized through caveolae endocytosis, which was later demonstrated to be a technical error and only induced by crosslinking of GPI-APs with primary and secondary antibodies [Mayor et al., 1994]. The clustering-induced caveolae
sequestration of GPI-APs was confirmed in several other studies [Fujimoto, 1996; Parton et al., 1994]. Caveolae are abundant cell surface organelles that can be endocytosed [Hill et al., 2008].
However, caveolae are described as highly stable and immobile plasma membrane domains that are not involved in constitutive endocytosis [Thomsen et al., 2002]. It is hypothesized though, that endocytosis of caveolae can be triggered e.g. by SV40 sequestered into caveolae [Pelkmans et al., 2002].
The current model suggests that caveolae biogenesis starts at the Golgi by caveolin
oligomerization and cholesterol binding. At the plasma membrane, polymerase I and transcript release factor (PTRF)-Cavin (also cavin-1) is recruited to caveolae and is suggested to form multiprotein complexes with other cavins and to operate as coat protein [Hill et al. 2008;
Bastiani et al., 2009]. Binding of PTRF-Cavin to caveolae stabilizes the membrane curvature to produce the classic 50-80 nm flask shape of mature caveolae.
Various GPI-APs are internalized primarily by the CLIC/GEEC pathway (Fig.4) [Sabharanjak et al., 2002; Chadda et al., 2007; Kumari et al., 2008]. The CLIC/GEEC pathway is an endocytic process that is independent of clathrin and dynamin and is regulated by the small GTPases arf1 and cdc42 [Chadda et al., 2007; Kumari et al., 2008]. The morphologically distinct CLICs were identified as the primary carriers that form directly from the plasma membrane and mature into GEECs and finally merge with common early endosomes (EEs) [Kirkham et al., 2005; Kalia et al., 2006]. This pathway was identified as a high capacity route that is proposed to account for the bulk of constitutive fluid uptake and is implicated as a key mediator of plasma membrane remodeling and maintenance [Howes et al., 2010]. The 3D structural analysis identifies CLICs as pleiomorphic, complex, multicomponent carriers. GEECs can be described as acidic fluid-
containing endosomes carrying specialized membrane cargo that undergo heterotypic fusion with EE and potentially homotypic fusion with other GEECs involving the activation of the GTPase Rab5 and the recruitment of early endosome antigen-1 (EEA1) [Kalia et al., 2006]. The pH of GEECs is remarkably acidic at 6.0 and implies an efficient, however, yet uncharacterized acidification mechanism, presumably requiring the V-ATPase like other acidic organelles. The Rho GTPase-activating protein (GAP) domain-containing protein GRAF1 was shown to be required for the CLIC/GEEC pathway [Lundmark et al., 2008]. GRAF1 localizes to PI4,5P
2- enriched, tubular and punctate lipid structures through its N-terminal BAR and PH domains.
These membrane carriers are devoid of caveolin-1 and flotillin 1, but are associated with cdc42
18 activity. Studies on the endocytosis of non-crosslinked FR using a monovalent fluorescent
analogue of folic acid show its internalization through the CLIC/GEEC pathway. FR as well as other GPI-APs are believed to cycle between the plasma membrane and endosomal
compartments [Kamen at al., 1988]. At early time points after internalization, FR was shown to localize to GEECs which are distinct from endosomes containing endocytosed transferrin receptor (TfR). The two endosomal subpopulations then fuse with each other and merge with recycling endosomes (REs) [Sabharanjak et al., 2002]. The replacement of the GPI-anchor of FR with a TM domain resulted in internalization through CME, however less efficiently [Ritter et al., 1995]. Endocytosis of FR into CLICs/GEECs is regulated by the GTPase cdc42, shown by
overexpression of a dominant-negative form of cdc42. CME of TfR was unaffected under these conditions [Sabharanjak et al., 2002]. Cholesterol depletion resulted in dramatic redirection of FR into TfR-containing endosomes which suggests that the association of GPI-APs with ordered lipid domains might be a determinant for their internalization through this pathway.
However, some GPI-APs were found to be internalized through different pathways [Kalia et al., 2006]. In HeLa cells, CD59 was found in arf6-associated endosomes together with major histocompatibility protein class I (MHC I). Endocytosis of CD59 and MHC I required cholesterol as it was inhibited by filipin binding to the cell surface. Overexpression of active arf6 stimulated endocytosis of CD59 and MHC I to a similar extent, whereas transferrin (Tf) was not affected [Naslavsky et al., 2004]. In contrast, in CHO cells, activation or inactivation of arf6 did not
significantly change the uptake of GPI-APs [Kalia et al., 2006]. It seems that in these cells GPI-APs are constitutively endocytosed by an ar6-independent mechanism. It is possible that, although arf6 is present in CHO cells and can localize to Tf-positive EEs, the arf6-dependent endocytosis machinery is suppressed or that CHO cells lack an essential component of this transport machinery [Kalia et al., 2006].
In polarized hepatic cells, basolateral internalization of GPI-APs was shown to be dependent on flotillin-2 and dynamin, but not on cdc42 [Aït-Slimane et al., 2009].
Furthermore, other GPI-APs such as urokinase type plasminogen activator receptor (uPAR) and
PrP have been suggested to be internalized through CME, likely directed by their association
with transmembrane proteins [Czekay et al., 2001; Magalhães et al., 2002].
19 Recycling of GPI-anchored proteins
The CLIC/GEEC pathway internalizes and returns significant portions of the plasma membrane quickly, rapidly turning over membrane during key cellular processes such as plasma membrane repair and maintenance. Quantitation of endocytosed dextran demonstrated that up to 40% of endocytosed fluid, mainly internalized by this pathway, is regurgitated in 5 minutes [Chadda et al., 2007]. This provides a mechanism for quick plasma membrane remodeling required for various processes such as adhesion or migration [Howes et al., 2010]. It has previously been shown that recycling from a GPI-AP-positive endocytic compartment provides a significant fraction of required membrane during cell spreading [Gauthier et al., 2009]. Macropinocytosis, a pathway that mediates bulk update of membranes, fluid and signaling receptors, was also
suggested to participate in the very rapid turnover of membrane components that are critical for stimulated cell migration [Gu et al., 2011]. However, the rapid recycling route from CLIC/GEEC compartments to the plasma membrane is believed to occur prior to the fusion with EEs and provides an additional pathway distinct from the classical recycling route through REs.
The majority of endocytosed membrane internalized by the CLIC/GEEC pathway presumably fuses with EEs [Howes et al., 2010]. Subpopulations of EEs destined for recycling fuse with REs [Sabharanjak et al., 2002]. Recycling of GPI-APs from REs back to the plasma membrane has been shown to be affected by sphingolipid and cholesterol depletion [Chatterjee et al., 2001;
Mayor et al., 1998]. Most GPI-APs are recycled back to the plasma membrane at a rate at least 3- fold slower than recycling receptors such as TfR. The replacement of the GPI-anchor with a TM domain abolished the retention in REs [Chatterjee et al., 2001]. Cholesterol or sphingolipid depletion specifically enhanced the recycling rate to that of recycling receptors [Chatterjee et al., 2001; Mayor et al., 1998].
Project goal
We are particularly interested in the intracellular transport of GPI-APs in mammalian cells which is a fascinating topic for several reasons. First of all, as described earlier, GPI-APs lack a cytosolic domain and can therefore not be directly recognized by transport machineries localized in the cytosol, which raises the very important question about possible concentration and sorting mechanisms that enable efficient packaging of GPI-APs into transport carriers.
Secondly, GPI-APs consist of a protein, a glycan and a lipid part which creates the potential for a
variety of interactions that can determine or drive intracellular transport processes, not only
protein-protein interactions, but also protein-glycan or lipid-lipid interactions. Thirdly, GPI
20 biogenesis is essential for the embryonic development in mammals, which makes GPI-APs a very important protein class. In my opinion this indicates a high probability for the existence of precisely regulated transport mechanisms to ensure the proper expression of GPI-APs.
In this study, we investigate the intracellular transport of GPI-APs including the ER-to-Golgi transport, Golgi-to-plasma membrane transport and endocytosis in CHO cells, a non-polarized mammalian cell line. We particularly focus on the involvement and the importance of GPI anchor remodeling in these intracellular transport events and take advantage of GPI anchor remodeling mutants. The major part of this thesis addresses the long-standing debate about the putative importance of ordered lipid domains for GPI-AP sorting in the secretory and endocytic
pathways. By using lipid remodeling mutant cells expressing exclusively unremodeled GPI-APs that carry an unsaturated fatty acid in their lipid moiety and fail to associate with ordered lipid domains, we provide a new tool to challenge the hypothesis of lipid-mediated sorting of GPI-APs.
Furthermore, we established two novel quantitative transport assays with the SNAP-tag labeling
technology to measure the kinetics of Golgi-to-plasma membrane transport and endocytosis of
GPI-APs which provide powerful tools that can be applied for future investigations in the field of
protein trafficking.
21
II Results I
Manuscript:
Exit of GPI-anchored proteins from the ER differs in yeast and mammalian cells
Rivier et al, 2010 (Traffic)
22
In this study, we examined the ER exit of GPI-anchored proteins and analyzed the differences
between yeast and mammalian cells. My contributions to this work are minor and unfortunately
my data did not make it into the manuscript.
Traffic 2010;11:1017–1033 ©2010 John Wiley & Sons A/S doi:10.1111/j.1600-0854.2010.01081.x
Exit of GPI-Anchored Proteins from the ER Differs in Yeast and Mammalian Cells
Anne-Sophie Rivier1, Guillaume A. Castillon1, Laetitia Michon1, Masayoshi Fukasawa2, Maria Romanova-Michaelides1, Nina Jaensch1, Kentaro Hanada2and Reika Watanabe1,∗
1Department of Biochemistry, University of Geneva, Sciences II, 30 quai Ernest-Ansermet, CH-1211 Geneva, Switzerland
2Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640, Japan
*Corresponding author: Reika Watanabe, reika.watanabe@unige.ch
Previous studies have shown that yeast glycosylphos- phatidylinositol-anchored proteins (GPI-APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI-APs into COPII vesicles requires specific lipid interactions.
We compared the ER exit mechanism and segregation of GPI-APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER-to-Golgi transport of GPI-APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI-APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI-APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI-APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.
Key words: COPII, detergent-resistant membrane, ER exit site, GPI-anchored protein, lipid raft, protein sorting, Sar1 protein, Sphingolipid
Received 13 October 2009, revised and accepted for pub- lication 10 May 2010, uncorrected manuscript published online 11 May 2010
Secretory proteins are synthesized on ribosomes bound to the endoplasmic reticulum (ER) and inserted co-translationally into the ER membrane, from where they are transported via the Golgi apparatus to their final destination on the cell surface or in other intracellular membrane compartments. Export of proteins from the ER is coordinated by assembly of coat protein complex II (COPII) at discrete sites on the ER surface known as ER exit sites (ERES). In the yeastSaccharomyces cerrevisiae
and in mammalian cells, assembly of the COPII coat is initiated by Sec12-dependent GDP–GTP exchange on the small GTPase Sar1 (1). The activated Sar1-GTP sub- sequently recruits the Sec23–Sec24 and Sec13–Sec31 complexes to the exit sites (1).
Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are another class of eukaryotic membrane protein, which are held in the membrane by a GPI lipid anchor.
GPI biosynthesis is essential in yeast, and defective biosynthesis in mammals causes embryonic lethality (2,3).
In contrast to other lipid anchors, the GPI anchor has a complex structure, including a conserved glycan core and a phospholipid tail. GPI anchors are synthesized from phosphatidylinositol (PI) and various sugar donors in the ER (4,5). The precursor proteins have an N-terminal signal peptide and a GPI-attachment signal at the C-terminus.
This GPI-attachment signal is subsequently cleaved and a preassembled GPI anchor is transferred in the ER. Without GPI attachment, the precursor proteins are not expressed at the cell surface but instead accumulate mainly in the ER (6–8).
The lipid moieties of GPI-APs are sequentially remodeled both in yeast and mammalian cells. The proteins respon- sible for lipid remodeling were cloned recently from yeast and mammals (9–12). They show significant homologies to each other, indicating the likely conserved biological role of lipid remodeling of the GPI anchor (4,5,13). Inter- estingly, the yeast remodeling enzymes are located in the ER, whereas the mammalian proteins are mainly in the Golgi apparatus (10–12), suggesting that remodel- ing occurs in different membrane compartments in yeast and mammalian cells. We do not fully understand the functional consequences of lipid remodeling of GPI-APs;
however, remodeling confers on GPI-APs the property to co-purify with the detergent-resistant membrane (DRM) fraction (9,10,12). This association with DRMs has been postulated to reflect the incorporation of GPI-APs into lipid rafts, which contain mainly sphingolipids and sterols (14).
Incorporation into lipid rafts may target the GPI-APs to par- ticular regions of the cell. In polarized mammalian epithelial cells, e.g., most GPI-APs are preferentially targeted to the apical domain of the plasma membrane.
In yeast, GPI-APs and other secretory proteins are prefer- entially transported from the ER to the Golgi apparatus in different vesicle populations (15–17). Consistent with this, Castillon et al. (18) observed, by using thermosensitive mutantsec31-1 yeast cells that accumulate cargo proteins in the ERES, that GPI-APs accumulate in ERES that are dis- tinct from those in which other secretory proteins accumu- late. Furthermore, in these cells, concentration of GPI-APs www.traffic.dk 1017
