Grd19/Snx3p functions as a cargo-specific adapter for retromer-dependent endocytic recycling

Amajor function of the endocytic system is the sorting of cargo to various organelles. Endocytic sorting of the yeast reductive iron transporter, which is composed of the Fet3 and Ftr1 proteins, is regulated by available iron. When iron is provided to iron-starved cells, Fet3p–Ftr1p is targeted to the lysosome-like vacuole and degraded. In contrast, when iron is not available, Fet3p–Ftr1p is maintained on the plasma membrane via an endocytic recycling pathway requiring the sorting nexin Grd19/Snx3p, the pentameric retromer complex, and the Ypt6p Golgi Rab GTPase module. A recycling signal in Ftr1p was identified and found to bind directly to Grd19/Snx3p. Retromer and Grd19/Snx3p partially colocalize to tubular endosomes, where they are physically associated. After export from the endosome, Fet3p–Ftr1p transits through the Golgi apparatus for resecretion. Thus, Grd19/Snx3p, functions as a cargo-specific adapter for the retromer complex, establishing a precedent for a mechanism by which sorting nexins expand the repertoire of retromer-dependent cargos.

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Chimeric Forms of Furin and Tgn38 Are Transported from the Plasma Membrane to the Trans-Golgi Network via Distinct Endosomal Pathways

The Journal of Cell Biology ◽

10.1083/jcb.146.2.345 ◽

1999 ◽

Vol 146 (2) ◽

pp. 345-360 ◽

Cited By ~ 149

Author(s):

William G. Mallet ◽

Frederick R. Maxfield

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Chimeric Proteins ◽

Endocytic Recycling ◽

Single Pass ◽

Trans Golgi Network ◽

Late Endosomes ◽

Recycling Pathway ◽

Endosomal Transport ◽

Golgi Network

Furin and TGN38 are membrane proteins that cycle between the plasma membrane and the trans-Golgi network (TGN), each maintaining a predominant distribution in the TGN. We have used chimeric proteins with an extracellular Tac domain and the cytoplasmic domain of TGN38 or furin to study the trafficking of these proteins in endosomes. Previously, we demonstrated that the postendocytic trafficking of Tac-TGN38 to the TGN is via the endocytic recycling pathway (Ghosh, R.N., W.G. Mallet, T.T. Soe, T.E. McGraw, and F.R. Maxfield. 1998. J. Cell Biol. 142:923–936). Here we show that internalized Tac-furin is delivered to the TGN through late endosomes, bypassing the endocytic recycling compartment. The transport of Tac-furin from late endosomes to the TGN appears to proceed via an efficient, single-pass mechanism. Delivery of Tac-furin but not Tac-TGN38 to the TGN is blocked by nocodazole, and the two pathways are also differentially affected by wortmannin. These studies demonstrate the existence of two independent pathways for endosomal transport of proteins to the TGN from the plasma membrane.

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An Acidic Cluster in the Cytosolic Domain of Human Cytomegalovirus Glycoprotein B Is a Signal for Endocytosis from the Plasma Membrane

Journal of Virology ◽

10.1128/jvi.73.10.8677-8688.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8677-8688 ◽

Cited By ~ 42

Author(s):

Sharof Tugizov ◽

Ekaterina Maidji ◽

Jianqiao Xiao ◽

Lenore Pereira

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Human Cytomegalovirus ◽

Human Fibroblasts ◽

Endocytic Pathway ◽

Glycoprotein B ◽

Endocytic Recycling ◽

Cytosolic Domain ◽

Recycling Pathway ◽

Apical Membranes

ABSTRACT We previously reported that human cytomegalovirus (CMV) glycoprotein B (gB) is transported to apical membranes in CMV-infected polarized retinal pigment epithelial (ARPE-19) cells and in Madin-Darby canine kidney (MDCK) epithelial cells constitutively expressing gB. The cytosolic domain of gB contains a cluster of acidic amino acids, a motif that plays a pivotal role in vectorial trafficking in polarized epithelial cells and may also function as a signal for entry into the endocytic pathway. Here we compared gB internalization and recycling to the plasma membrane in CMV-infected human fibroblasts (HF) and ARPE-19 cells by using antibody-internalization experiments. Immunofluorescence and quantitative assays showed that gB was internalized from the cell surface into clathrin-coated transport vesicles and then recycled to the plasma membrane. gB colocalized with clathrin-coated vesicles containing the transferrin receptor in the early endocytic/recycling pathway, indicating that gB traffics in this pathway. The specific role of the acidic cluster in regulating the sorting of gB-containing vesicles in the early endocytic/recycling pathway was examined in MDCK cells expressing mutated gB derivatives. Immunofluorescence assays showed that derivatives lacking the acidic cluster were impaired in internalization and failed to recycle. These findings, together with our earlier observation that the acidic cluster is a key determinant for targeting gB molecules to apical membranes in epithelial cells, establish that this signal is recognized by cellular proteins that participate in polarized sorting and transport in the early endocytic/recycling pathway.

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Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721760115 ◽

2018 ◽

Vol 115 (14) ◽

pp. 3716-3721 ◽

Cited By ~ 24

Author(s):

Yuliya Salanenka ◽

Inge Verstraeten ◽

Christian Löfke ◽

Kaori Tabata ◽

Satoshi Naramoto ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Protein Trafficking ◽

Molecular Mechanisms ◽

Sorting Nexin ◽

Protein Traffic ◽

Della Proteins ◽

Retromer Complex ◽

Ga Signaling ◽

Multiple Processes

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses.

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Retromer and sorting nexins in endosomal sorting

Biochemical Society Transactions ◽

10.1042/bst20140290 ◽

2015 ◽

Vol 43 (1) ◽

pp. 33-47 ◽

Cited By ~ 108

Author(s):

Matthew Gallon ◽

Peter J. Cullen

Keyword(s):

Plasma Membrane ◽

Present Article ◽

Transmembrane Proteins ◽

Molecular Architecture ◽

Sorting Nexins ◽

Endosomal Sorting ◽

Sorting Nexin ◽

Cargo Proteins ◽

Associated Proteins ◽

Lysosome Related Organelles

The evolutionarily conserved endosomal retromer complex rescues transmembrane proteins from the lysosomal degradative pathway and facilitates their recycling to other cellular compartments. Retromer functions in conjunction with numerous associated proteins, including select members of the sorting nexin (SNX) family. In the present article, we review the molecular architecture and cellular roles of retromer and its various functional partners. The endosomal network is a crucial hub in the trafficking of proteins through the cellular endomembrane system. Transmembrane proteins, here termed cargos, enter endosomes by endocytosis from the plasma membrane or by trafficking from the trans-Golgi network (TGN). Endosomal cargo proteins face one of the two fates: retention in the endosome, leading ultimately to lysosomal degradation or export from the endosome for reuse (‘recycling’). The balance of protein degradation and recycling is crucial to cellular homoeostasis; inappropriate sorting of proteins to either fate leads to cellular dysfunction. Retromer is an endosome-membrane-associated protein complex central to the recycling of many cargo proteins from endosomes, both to the TGN and the plasma membrane (and other specialized compartments, e.g. lysosome-related organelles). Retromer function is reliant on a number of proteins from the SNX family. In the present article, we discuss this inter-relationship and how defects in retromer function are increasingly being linked with human disease.

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The rab Exchange Factor Sec2p Reversibly Associates with the Exocyst

Molecular Biology of the Cell ◽

10.1091/mbc.e05-10-0917 ◽

2006 ◽

Vol 17 (6) ◽

pp. 2757-2769 ◽

Cited By ~ 52

Author(s):

Martina Medkova ◽

Y. Ellen France ◽

Jeff Coleman ◽

Peter Novick

Keyword(s):

Plasma Membrane ◽

Small Region ◽

Rab Gtpase ◽

Temperature Sensitive ◽

Secretory Vesicles ◽

Nucleotide Exchange ◽

Vesicle Tethering ◽

Exchange Factor ◽

Polarized Transport ◽

Recycling Pathway

Activation of the rab GTPase, Sec4p, by its exchange factor, Sec2p, is needed for polarized transport of secretory vesicles to exocytic sites and for exocytosis. A small region in the C-terminal half of Sec2p regulates its localization. Loss of this region results in temperature-sensitive growth and the depolarized accumulation of secretory vesicles. Here, we show that Sec2p associates with the exocyst, an octameric effector of Sec4p involved in tethering secretory vesicles to the plasma membrane. Specifically, the exocyst subunit Sec15p directly interacts with Sec2p. This interaction normally occurs on secretory vesicles and serves to couple nucleotide exchange on Sec4p to the recruitment of the Sec4p effector. The mislocalization of Sec2p mutants correlates with dramatically enhanced binding to the exocyst complex. We propose that Sec2p is normally released from the exocyst after vesicle tethering so that it can recycle onto a new round of vesicles. The mislocalization of Sec2p mutants results from a failure to be released from Sec15p, blocking this recycling pathway.

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Ascorbic acid increases SVCT2 localization at the plasma membrane by accelerating its trafficking from early secretory compartments and through the endocytic-recycling pathway

Free Radical Biology and Medicine ◽

10.1016/j.freeradbiomed.2018.03.013 ◽

2018 ◽

Vol 120 ◽

pp. 181-191 ◽

Cited By ~ 4

Author(s):

A. Covarrubias-Pinto ◽

A.I. Acuña ◽

G. Boncompain ◽

E. Papic ◽

P.V. Burgos ◽

...

Keyword(s):

Ascorbic Acid ◽

Plasma Membrane ◽

Endocytic Recycling ◽

Recycling Pathway

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Sorting Nexin 17 Accelerates Internalization Yet Retards Degradation of P-selectin

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0143 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3095-3105 ◽

Cited By ~ 33

Author(s):

Ross Williams ◽

Thomas Schlüter ◽

Marnie S. Roberts ◽

Peter Knauth ◽

Ralf Bohnensack ◽

...

Keyword(s):

Endothelial Cells ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Cell Surface ◽

Mammalian Cells ◽

Tight Control ◽

Yeast Two Hybrid ◽

Sorting Nexin ◽

Key Steps ◽

Two Hybrid

The transient appearance of P-selectin on the surface of endothelial cells helps recruit leukocytes into sites of inflammation. The tight control of cell surface P-selectin on these cells depends on regulated exocytosis of Weibel-Palade bodies where the protein is stored and on its rapid endocytosis. After endocytosis, P-selectin is either sorted via endosomes and the Golgi apparatus for storage in Weibel-Palade bodies or targeted to lysosomes for degradation. A potential player in this complex endocytic itinerary is SNX17, a member of the sorting nexin family, which has been shown in a yeast two-hybrid assay to bind P-selectin. Here, we show that overexpression of SNX17 in mammalian cells can influence two key steps in the endocytic trafficking of P-selectin. First, it promotes the endocytosis of P-selectin from the plasma membrane. Second, it inhibits the movement of P-selectin into lysosomes, thereby reducing its degradation.

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SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways

The Journal of Cell Biology ◽

10.1083/jcb.201111121 ◽

2012 ◽

Vol 197 (2) ◽

pp. 219-230 ◽

Cited By ~ 114

Author(s):

Florian Steinberg ◽

Kate J. Heesom ◽

Mark D. Bass ◽

Peter J. Cullen

Keyword(s):

Isotope Labeling ◽

Degradation Pathway ◽

Lysosomal Degradation ◽

Sorting Nexins ◽

Retromer Complex ◽

Proteomic Approach ◽

Cytoplasmic Tails ◽

Npxy Motif ◽

Global Identification ◽

Insight Into

The FERM-like domain–containing sorting nexins of the SNX17/SNX27/SNX31 family have been proposed to mediate retrieval of transmembrane proteins from the lysosomal pathway. In this paper, we describe a stable isotope labeling with amino acids in culture–based quantitative proteomic approach that allows an unbiased, global identification of transmembrane cargoes that are rescued from lysosomal degradation by SNX17. This screen revealed that several integrins required SNX17 for their stability, as depletion of SNX17 led to a loss of β1 and β5 integrins and associated a subunits from HeLa cells as a result of increased lysosomal degradation. SNX17 bound to the membrane distal NPXY motif in β integrin cytoplasmic tails, thereby preventing lysosomal degradation of β integrins and their associated a subunits. Furthermore, SNX17-dependent retrieval of integrins did not depend on the retromer complex. Consistent with an effect on integrin recycling, depletion of SNX17 also caused alterations in cell migration. Our data provide mechanistic insight into the retrieval of internalized integrins from the lysosomal degradation pathway, a prerequisite for subsequent recycling of these matrix receptors.

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Membrane Targeting Properties of a Herpesvirus Tegument Protein-Retrovirus Gag Chimera

Journal of Virology ◽

10.1128/jvi.74.18.8692-8699.2000 ◽

2000 ◽

Vol 74 (18) ◽

pp. 8692-8699 ◽

Cited By ~ 38

Author(s):

J. Bradford Bowzard ◽

Robert J. Visalli ◽

Carol B. Wilson ◽

Joshua S. Loomis ◽

Eric M. Callahan ◽

...

Keyword(s):

Amino Acids ◽

Plasma Membrane ◽

Golgi Apparatus ◽

Solution Structure ◽

Chimeric Protein ◽

Membrane Targeting ◽

Heterologous Proteins ◽

Gag Protein ◽

Internal Membrane ◽

Simplex Virus

ABSTRACT The retroviral Gag protein is capable of directing the production and release of virus-like particles in the absence of all other viral components. Budding normally occurs after Gag is transported to the plasma membrane by its membrane-targeting and -binding (M) domain. In the Rous sarcoma virus (RSV) Gag protein, the M domain is contained within the first 86 amino acids. When M is deleted, membrane association and budding fail to occur. Budding is restored when M is replaced with foreign membrane-binding sequences, such as that of the Src oncoprotein. Moreover, the RSV M domain is capable of targeting heterologous proteins to the plasma membrane. Although the solution structure of the RSV M domain has been determined, the mechanism by which M specifically targets Gag to the plasma membrane rather than to one or more of the large number of internal membrane surfaces (e.g., the Golgi apparatus, endoplasmic reticulum, and nuclear, mitochondrial, or lysosomal membranes) is unknown. To further investigate the requirements for targeting proteins to discrete cellular locations, we have replaced the M domain of RSV with the product of the unique long region 11 (UL11) gene of herpes simplex virus type 1. This 96-amino-acid myristylated protein is thought to be involved in virion transport and envelopment at internal membrane sites. When the first 100 amino acids of RSV Gag (including the M domain) were replaced by the entire UL11 sequence, the chimeric protein localized at and budded into the Golgi apparatus rather than being targeted to the plasma membrane. Myristate was found to be required for this specific targeting, as were the first 49 amino acids of UL11, which contain an acidic cluster motif. In addition to shedding new light on UL11, these experiments demonstrate that RSV Gag can be directed to internal cellular membranes and suggest that regions outside of the M domain do not contain a dominant plasma membrane-targeting motif.

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