Ent3p Is a PtdIns(3,5)P2 Effector Required for Protein Sorting to the Multivesicular Body

Friant, Sylvie; Pécheur, Eve Isabelle; Eugster, Anne; Michel, Fabrice; Lefkir, Yaya; Nourrisson, Delphine; Letourneur, François

doi:10.1016/s1534-5807(03)00238-7

Cited by 114 publications

(137 citation statements)

References 47 publications

(4 reference statements)

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“…In a similar vein to that described for the FAPP1 PH domain and PtdIns4P, we have been unable, with the vesicle-based techniques described above, to detect PtdIns(3,5)P 2 specificity for two interesting classes of protein: mammalian Vps24p [22] (a component of the ESCRT III complex), and Ent3p/Ent5p from S. cerevisiae [11,68]. In both of these cases, the primary evidence in support of PtdIns(3,5)P 2 specificity came from studies in which the phosphoinositide was studied in isolation.…”

Section: Ptdins(35)p 2 -Specific Binding Proteinsmentioning

confidence: 57%

“…Phage display was used to identify mVps24p as a protein that binds to immobilized biotinylated PtdIns(3,5)P 2 [22]. Lipid overlay/dot-blot approaches provided the initial suggestion that the ENTH domains of Ent3p and Ent5p bind PtdIns(3,5)P 2 [11,68]. Both sets of studies also employed vesicle sedimentation approaches using 5% (mole/mole) phosphoinositide in a reasonable lipid background.…”

Section: Ptdins(35)p 2 -Specific Binding Proteinsmentioning

confidence: 99%

“…Of the 13 most populous classes of signaling interaction domain found in the human proteome [3], members of more than half have been reported to drive reversible membrane association by such interactions [1,2,4,5]. Domains that have been implicated in head group-specific recognition of phosphoinositides include pleckstrin homology (PH) domains [6,7]; phagocyte oxidase (phox) homology (PX) domains [8]; FYVE domains (for Fab1, YOTB, Vac1 and EEA1) [9]; epsin or AP180 N-terminal homology (ENTH/ANTH) domains [10,11]; and plant homeodomain (PHD) zinc fingers [12]. In addition, phosphoinositide binding has been reported for PDZ (for Postsynaptic density protein, Disc large, Zona occludens) domains [13], FERM (for band Four-point-one, Ezrin, Radixin, Moesin) domains [14], Tubby [15], and MARCKS [16] proteins.…”

Section: Introductionmentioning

confidence: 99%

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Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

Methods

119

121

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The burgeoning of phosphoinositide-binding domains and proteins in cellular signaling and trafficking has drawn laboratories from a wide variety of fields into the study of lipid interactions with peripheral membrane proteins. Many different approaches have been developed to assess phosphoinositide binding, some of which are more problematic than others, and some of which can be quantitated more readily than others. With a focus on the methods used in our laboratory, we describe here the considerations that need to be taken into account when establishing -and quantitating -the specific binding of a protein or domain to phosphoinositides in membranes. We also discuss briefly a few examples in which no clear consensus has yet been reached as to the specificity of a given domain or protein because of discrepancies between different commonlyused approaches.

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Section: Ptdins(35)p 2 -Specific Binding Proteinsmentioning

confidence: 57%

Section: Ptdins(35)p 2 -Specific Binding Proteinsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Determining selectivity of phosphoinositide-binding domains

Narayan

Lemmon

2006

Methods

119

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“…A single ent3⌬ deletion strain does not display the defects in MVB sorting observed in ent3-1 mutant cells. This suggested that the point mutation in the ENTH domain of Ent3p found in ent3-1 mutant cells had a dominant effect on MVB protein sorting and that there could be additional PtdIns(3,5)P 2 effectors required for MVB sorting (Friant et al, 2003). In our query for ENTH domain proteins redundant to Ent3p, we identified Ent5p.…”

mentioning

confidence: 96%

“…The ENTH domain promotes membrane recruitment by binding phosphoinositides . We have recently reported that Ent3p is a specific PtdIns(3,5)P 2 effector involved in protein sorting at the MVB in yeast (Friant et al, 2003). Analysis of yeast mutants unable to synthesize PtdIns(3,5)P 2 , such as fab1, vac7, and vac14, revealed that this phospholipid is also required for sorting ubiquitinated cargoes into the MVB (Bonangelino et al, 1997;Odorizzi et al, 1998;Reggiori and Pelham, 2001;Dove et al, 2002).…”

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confidence: 99%

Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

Eugster¹,

Pécheur²,

Michel

et al. 2004

MBoC

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At the late endosomes, cargoes destined for the interior of the vacuole are sorted into invaginating vesicles of the multivesicular body. Both PtdIns(3,5)P 2 and ubiquitin are necessary for proper sorting of some of these cargoes. We show that Ent5p, a yeast protein of the epsin family homologous to Ent3p, localizes to endosomes and specifically binds to PtdIns(3,5)P 2 via its ENTH domain. In cells lacking Ent3p and Ent5p, ubiquitin-dependent sorting of biosynthetic and endocytic cargo into the multivesicular body is disrupted, whereas other trafficking routes to the vacuole are not affected. Ent3p and Ent5p are associated with Vps27p, a FYVE domain containing protein that interacts with ubiquitinated cargoes and is required for protein sorting into the multivesicular body. Therefore, Ent3p and Ent5p are the first proteins shown to be connectors between PtdIns(3,5)P 2 -and the Vps27p-ubiquitin-driven sorting machinery at the multivesicular body. INTRODUCTIONEndosomes are crossroads between the biosynthetic and the endocytic pathways. Here, proteins destined for the lysosome/vacuole are segregated away from proteins recycling back to the plasma membrane or to the Golgi apparatus. A critical membrane segregation and protein-sorting event takes place in late endosomes, regions of endosomal membranes invaginate and vesicles bud into the lumen of the organelle, giving rise to the multivesicular body (MVB;Felder et al., 1990). Mature MVB fuses with the vacuole/ lysosome releasing internal vesicles into its lumen and thereby exposing them to the activity of hydrolytic enzymes (Futter et al., 1996;Odorizzi et al., 1998). This mechanism allows a subset of endosomal membrane proteins to be sorted to the lumen of the lysosome/vacuole, whereas others are either selectively recycled back to the Golgi or to the plasma membrane, or they end up on the lysosomal/vacuolar membrane (Pelham, 2002). In yeast, the MVB pathway is crucial for the regulated turnover of pheromone receptors and of some permeases (Odorizzi et al., 1998). Further, biosynthetic transmembrane proteins such as carboxypeptidase S (Cps1p) and Phm5p follow this route to reach their final destination, the vacuolar lumen (Odorizzi et al., 1998;Reggiori and Pelham, 2001;Epple et al., 2003).Ubiquitination has been implicated as an endosomal-sorting signal. Cps1p and Phm5p ubiquitination is essential to target them into the vacuolar lumen after sorting at the MVB (Katzmann et al., 2001;Reggiori and Pelham, 2001). Potential cargo receptors responsible for recognition and recruitment of ubiquitinated cargo into MVB vesicles are class E Vps (vacuolar protein sorting) proteins. Class E mutants accumulate cargo destined for the vacuole in an exaggerated endosomal structure and all (currently identified) class E proteins are required for sorting at the MVB (Conibear and Stevens, 1998). Vps23p, a class E protein of the ESCRT-I complex (endosomal-sorting complex required for transport), carries an ubiquitin-conjugating (UBC)-like domain and interacts directly with ubiquitin i...

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Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

Balla

Várnai

2009

CP Cell Biology

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This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present in only tiny amounts as compared to structural lipids, but they are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane recruitment and activity of many membrane protein signaling complexes in specific membrane compartments, and they have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single‐cell level. The only available technique in live cell applications is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules, when fused to fluorescent proteins, can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular localization, and it has rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. This unit summarizes the design and practical use of these constructs and also reviews important considerations for interpretation of the data obtained by this technique. Curr. Protoc. Cell Biol. 42:24.4.1‐24.4.27. © 2009 by John Wiley & Sons, Inc.

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Ent3p Is a PtdIns(3,5)P2 Effector Required for Protein Sorting to the Multivesicular Body

Cited by 114 publications

References 47 publications

Determining selectivity of phosphoinositide-binding domains

Determining selectivity of phosphoinositide-binding domains

Ent5p Is Required with Ent3p and Vps27p for Ubiquitin-dependent Protein Sorting into the Multivesicular Body

Visualization of Cellular Phosphoinositide Pools with GFP‐Fused Protein‐Domains

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