Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

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Interchangeable but Essential Functions of SNX1 and SNX2 in the Association of Retromer with Endosomes and the Trafficking of Mannose 6-Phosphate Receptors

Molecular and Cellular Biology ◽

10.1128/mcb.00156-06 ◽

2006 ◽

Vol 27 (3) ◽

pp. 1112-1124 ◽

Cited By ~ 140

Author(s):

Raul Rojas ◽

Satoshi Kametaka ◽

Carol R. Haft ◽

Juan S. Bonifacino

Keyword(s):

Mammalian Cells ◽

Human Cell Line ◽

Sorting Nexin ◽

Retromer Complex ◽

Cell Line Hela ◽

Endosomal Membranes ◽

Mannose 6 Phosphate ◽

And Function ◽

Golgi Network ◽

Functional Analyses

ABSTRACT The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.

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Cargo-selective SNX-BAR proteins mediate retromer trimer independent retrograde transport

The Journal of Cell Biology ◽

10.1083/jcb.201702137 ◽

2017 ◽

Vol 216 (11) ◽

pp. 3677-3693 ◽

Cited By ~ 60

Author(s):

Arunas Kvainickas ◽

Ana Jimenez-Orgaz ◽

Heike Nägele ◽

Zehan Hu ◽

Jörn Dengjel ◽

...

Keyword(s):

Quantitative Proteomics ◽

Retrograde Transport ◽

Dependent Manner ◽

Sorting Nexin ◽

Trans Golgi Network ◽

The Core ◽

Retromer Complex ◽

Mannose 6 Phosphate ◽

Golgi Network

The retromer complex, which recycles the cation-independent mannose 6-phosphate receptor (CI-MPR) from endosomes to the trans-Golgi network (TGN), is thought to consist of a cargo-selective VPS26–VPS29–VPS35 trimer and a membrane-deforming subunit of sorting nexin (SNX)–Bin, Amphyphysin, and Rvs (BAR; SNX-BAR) proteins. In this study, we demonstrate that heterodimers of the SNX-BAR proteins, SNX1, SNX2, SNX5, and SNX6, are the cargo-selective elements that mediate the retrograde transport of CI-MPR from endosomes to the TGN independently of the core retromer trimer. Using quantitative proteomics, we also identify the IGF1R, among more potential cargo, as another SNX5 and SNX6 binding receptor that recycles through SNX-BAR heterodimers, but not via the retromer trimer, in a ligand- and activation-dependent manner. Overall, our data redefine the mechanics of retromer-based sorting and call into question whether retromer indeed functions as a complex of SNX-BAR proteins and the VPS26–VPS29–VPS35 trimer.

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Rabenosyn-5, a Novel Rab5 Effector, Is Complexed with Hvps45 and Recruited to Endosomes through a Fyve Finger Domain

The Journal of Cell Biology ◽

10.1083/jcb.151.3.601 ◽

2000 ◽

Vol 151 (3) ◽

pp. 601-612 ◽

Cited By ~ 222

Author(s):

Erik Nielsen ◽

Savvas Christoforidis ◽

Sandrine Uttenweiler-Joseph ◽

Marta Miaczynska ◽

Frederique Dewitte ◽

...

Keyword(s):

Early Endosome ◽

Effector Proteins ◽

Endosomal Trafficking ◽

Membrane Domains ◽

Phosphatidylinositol 3 Kinase ◽

Early Endosomes ◽

Site Of Action ◽

Endosomal Membranes ◽

Novel Protein ◽

Phosphatidylinositol 3

Rab5 regulates endocytic membrane traffic by specifically recruiting cytosolic effector proteins to their site of action on early endosomal membranes. We have characterized a new Rab5 effector complex involved in endosomal fusion events. This complex includes a novel protein, Rabenosyn-5, which, like the previously characterized Rab5 effector early endosome antigen 1 (EEA1), contains an FYVE finger domain and is recruited in a phosphatidylinositol-3-kinase–dependent fashion to early endosomes. Rabenosyn-5 is complexed to the Sec1-like protein hVPS45. hVPS45 does not interact directly with Rab5, therefore Rabenosyn-5 serves as a molecular link between hVPS45 and the Rab5 GTPase. This property suggests that Rabenosyn-5 is a closer mammalian functional homologue of yeast Vac1p than EEA1. Furthermore, although both EEA1 and Rabenosyn-5 are required for early endosomal fusion, only overexpression of Rabenosyn-5 inhibits cathepsin D processing, suggesting that the two proteins play distinct roles in endosomal trafficking. We propose that Rab5-dependent formation of membrane domains enriched in phosphatidylinositol-3-phosphate has evolved as a mechanism for the recruitment of multiple effector proteins to mammalian early endosomes, and that these domains are multifunctional, depending on the differing activities of the effector proteins recruited.

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Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7677 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7677-7688 ◽

Cited By ~ 171

Author(s):

P Hu ◽

A Mondino ◽

E Y Skolnik ◽

J Schlessinger

Keyword(s):

Cell Cycle Progression ◽

Phosphatidylinositol 3 Kinase ◽

Intact Cells ◽

Src Homology 2 ◽

Genes Encoding ◽

Src Homology ◽

Acid Domain ◽

Vacuolar Protein ◽

Phosphatidylinositol 3

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

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Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

Molecular Biology of the Cell ◽

10.1091/mbc.e08-01-0080 ◽

2008 ◽

Vol 19 (12) ◽

pp. 5360-5372 ◽

Cited By ~ 715

Author(s):

Eisuke Itakura ◽

Chieko Kishi ◽

Kinji Inoue ◽

Noboru Mizushima

Keyword(s):

Membrane Trafficking ◽

Mammalian Cells ◽

Coiled Coil ◽

Pi3 Kinase ◽

Beclin 1 ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Interacting Protein ◽

Vacuolar Protein ◽

Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

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A Novel Mechanism for Localizing Membrane Proteins to YeastTrans-Golgi Network Requires Function of Synaptojanin-like Protein

Molecular Biology of the Cell ◽

10.1091/mbc.12.10.3175 ◽

2001 ◽

Vol 12 (10) ◽

pp. 3175-3190 ◽

Cited By ~ 21

Author(s):

Seon-Ah Ha ◽

Jeremy T. Bunch ◽

Hiroko Hama ◽

Daryll B. DeWald ◽

Steven F. Nothwehr

Keyword(s):

Membrane Proteins ◽

Secretory Pathway ◽

Protein A ◽

Retrograde Transport ◽

Cell Fractionation ◽

Gene Encoding ◽

Phosphoinositide Phosphatase ◽

Endosomal Membranes ◽

Delivery Mechanism ◽

Golgi Network

Localization of resident membrane proteins to the yeasttrans-Golgi network (TGN) involves both their retrieval from a prevacuolar/endosomal compartment (PVC) and a “slow delivery” mechanism that inhibits their TGN-to-PVC transport. A screen for genes required for the slow delivery mechanism uncoveredINP53, a gene encoding a phosphoinositide phosphatase. A retrieval-defective model TGN protein, A(F→A)-ALP, was transported to the vacuole in inp53 mutants approximately threefold faster than in wild type. Inp53p appears to function in a process distinct from PVC retrieval because combining inp53 with mutations that block retrieval resulted in a much stronger phenotype than either mutation alone. In vps27 strains defective for both anterograde and retrograde transport out of the PVC, a loss of Inp53p function markedly accelerated the rate of transport of TGN residents A-ALP and Kex2p into the PVC. Inp53p function is cargo specific because a loss of Inp53p function had no effect on the rate of Vps10p transport to the PVC in vps27 cells. The rate of early secretory pathway transport appeared to be unaffected ininp53 mutants. Cell fractionation experiments suggested that Inp53p associates with Golgi or endosomal membranes. Taken together, these results suggest that a phosphoinositide signaling event regulates TGN-to-PVC transport of select cargo proteins.

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FgRab5 and FgRab7 are essential for endosomes biogenesis and non-redundantly recruit the retromer complex to the endosomes in Fusarium graminearum

Stress Biology ◽

10.1007/s44154-021-00020-3 ◽

2021 ◽

Vol 1 (1) ◽

Author(s):

Yakubu Saddeeq Abubakar ◽

Han Qiu ◽

Wenqin Fang ◽

Huawei Zheng ◽

Guodong Lu ◽

...

Keyword(s):

Fusarium Graminearum ◽

Retrograde Transport ◽

Small Gtpases ◽

Rab Proteins ◽

Rab Gtpases ◽

Wild Type ◽

Retromer Complex ◽

Cargo Proteins ◽

Endosomal Membranes ◽

In The Wild

AbstractThe retromer complex, composed of the cargo-selective complex (CSC) Vps35-Vps29-Vps26 in complex with the sorting nexin dimer Vps5-Vps17, mediates the sorting and retrograde transport of cargo proteins from the endosomes to the trans-Golgi network in eukaryotic cells. Rab proteins belong to the Ras superfamily of small GTPases and regulate many trafficking events including vesicle formation, budding, transport, tethering, docking and fusion with target membranes. Herein, we investigated the potential functional relationship between the retromer complex and the 11 Rab proteins that exist in Fusarium graminearum using genetic and high-resolution laser confocal microscopic approaches. We found that only FgRab5 (FgRab5A and FgRab5B) and FgRab7 associate with the retromer complex. Both FgVps35-GFP and FgVps17-GFP are mis-localized and appear diffused in the cytoplasm of ΔFgrab5A, ΔFgrab5B and ΔFgrab7 mutants as compared to their punctate localization within the endosomes of the wild-type. FgRab7 and FgRab5B were found to co-localize with the retromer on endosomal membranes. Most strikingly, we found that these three Rab GTPases are indispensable for endosome biogenesis as both early and late endosomes could not be detected in the cells of the mutants after FM4-64 staining of the cells, while they were very clearly seen in the wild-type PH-1. Furthermore, FgRab7 was found to recruit FgVps35 but not FgVps17 to the endosomal membranes, whereas FgRab5B recruits both FgVps35 and FgVps17 to the membranes. Thus, we conclude that the Rab proteins FgRab5A, FgRab5B and FgRab7 play critical roles in the biogenesis of endosomes and in regulating retromer-mediated trafficking in F. graminearum.

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Assortment of Phosphatidylinositol 3-Kinase Complexes—Atg14p Directs Association of Complex I to the Pre-autophagosomal Structure in Saccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e05-09-0841 ◽

2006 ◽

Vol 17 (4) ◽

pp. 1527-1539 ◽

Cited By ~ 151

Author(s):

Keisuke Obara ◽

Takayuki Sekito ◽

Yoshinori Ohsumi

Keyword(s):

Saccharomyces Cerevisiae ◽

Complex I ◽

Regulatory Subunit ◽

Biological Processes ◽

Phosphatidylinositol 3 Kinase ◽

Yeast Saccharomyces Cerevisiae ◽

Complex Ii ◽

Vacuolar Protein ◽

Vacuolar Membranes ◽

Phosphatidylinositol 3

In the yeast Saccharomyces cerevisiae, two similar phosphatidylinositol 3-kinase complexes (complexes I and II) function in distinct biological processes, complex I in autophagy and complex II in the vacuolar protein sorting via endosomes. Atg14p is only integrated into complex I, likely facilitating the function of complex I in autophagy. Deletion analysis of Atg14p revealed that N-terminal region containing the coiled-coil structures was essential and sufficient for autophagy. Atg14p localized to pre-autophagosomal structure (PAS) and vacuolar membranes, whereas Vps38p, a component specific to complex II, localized to endosomes and vacuolar membranes. Vps34p and Vps30p, components shared by the two complexes, localized to the PAS, vacuolar membranes, and several punctate structures that included endosomes. The localization of these components to the PAS was Atg14p dependent but not dependent on Vps38p. Conversely, localization of these proteins to endosomes required Vps38p but not Atg14p. Vps15p, regulatory subunit of the Vps34p complexes, localized to the PAS, vacuolar membranes, and punctate structures independent of both Atg14p and Vps38p. Together, these results indicate that complexes I and II function in distinct biological processes by localizing to specific compartments in a manner mediated by specific components of each complex, Atg14p and Vps38p, respectively.

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Beclin–phosphatidylinositol 3‐kinase complex functions at the trans ‐Golgi network

EMBO Reports ◽

10.1093/embo-reports/kve061 ◽

2001 ◽

Vol 2 (4) ◽

pp. 330-335 ◽

Cited By ~ 587

Author(s):

Akio Kihara ◽

Yukiko Kabeya ◽

Yoshinori Ohsumi ◽

Tamotsu Yoshimori

Keyword(s):

Phosphatidylinositol 3 Kinase ◽

Trans Golgi Network ◽

Complex Functions ◽

Golgi Network ◽

Phosphatidylinositol 3

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Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603650113 ◽

2016 ◽

Vol 113 (29) ◽

pp. 8224-8229 ◽

Cited By ~ 34

Author(s):

Lindsey N. Young ◽

Kelvin Cho ◽

Rosalie Lawrence ◽

Roberto Zoncu ◽

James H. Hurley

Keyword(s):

Complex I ◽

Class Iii ◽

Phosphatidylinositol 3 Kinase ◽

Lipid Kinase ◽

Negative Stain ◽

Binding Factor ◽

Hydrogen Deuterium ◽

Negative Stain Electron Microscopy ◽

Vacuolar Protein ◽

Phosphatidylinositol 3

The class III phosphatidylinositol 3-kinase complex I (PI3KC3-C1) is central to autophagy initiation. We previously reported the V-shaped architecture of the four-subunit version of PI3KC3-C1 consisting of VPS (vacuolar protein sorting) 34, VPS15, BECN1 (Beclin 1), and ATG (autophagy-related) 14. Here we show that a putative fifth subunit, nuclear receptor binding factor 2 (NRBF2), is a tightly bound component of the complex that profoundly affects its activity and architecture. NRBF2 enhances the lipid kinase activity of the catalytic subunit, VPS34, by roughly 10-fold. We used hydrogen–deuterium exchange coupled to mass spectrometry and negative-stain electron microscopy to map NRBF2 to the base of the V-shaped complex. NRBF2 interacts primarily with the N termini of ATG14 and BECN1. We show that NRBF2 is a homodimer and drives the dimerization of the larger PI3KC3-C1 complex, with implications for the higher-order organization of the preautophagosomal structure.

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