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

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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Regulation of the endosomal SNX27-retromer by OTULIN

Nature Communications ◽

10.1038/s41467-019-12309-z ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Aurelia Stangl ◽

Paul R. Elliott ◽

Adan Pinto-Fernandez ◽

Sarah Bonham ◽

Luke Harrison ◽

...

Keyword(s):

Cell Surface ◽

Membrane Trafficking ◽

Pdz Domain ◽

Binding Motif ◽

Ubiquitin Chain ◽

Sorting Nexin ◽

Catalytic Function ◽

Retromer Complex ◽

Mass Spectrometric Identification ◽

Affinity Interaction

Abstract OTULIN (OTU Deubiquitinase With Linear Linkage Specificity) specifically hydrolyzes methionine1 (Met1)-linked ubiquitin chains conjugated by LUBAC (linear ubiquitin chain assembly complex). Here we report on the mass spectrometric identification of the OTULIN interactor SNX27 (sorting nexin 27), an adaptor of the endosomal retromer complex responsible for protein recycling to the cell surface. The C-terminal PDZ-binding motif (PDZbm) in OTULIN associates with the cargo-binding site in the PDZ domain of SNX27. By solving the structure of the OTU domain in complex with the PDZ domain, we demonstrate that a second interface contributes to the selective, high affinity interaction of OTULIN and SNX27. SNX27 does not affect OTULIN catalytic activity, OTULIN-LUBAC binding or Met1-linked ubiquitin chain homeostasis. However, via association, OTULIN antagonizes SNX27-dependent cargo loading, binding of SNX27 to the VPS26A-retromer subunit and endosome-to-plasma membrane trafficking. Thus, we define an additional, non-catalytic function of OTULIN in the regulation of SNX27-retromer assembly and recycling to the cell surface.

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Retromer is involved in epithelial Na+ channel trafficking

AJP Renal Physiology ◽

10.1152/ajprenal.00198.2019 ◽

2020 ◽

Vol 319 (5) ◽

pp. F895-F907 ◽

Cited By ~ 1

Author(s):

Tanya T. Cheung ◽

Anna C. Geda ◽

Adam W. Ware ◽

Sahib R. Rasulov ◽

Polly Tenci ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Early Endosome ◽

Na Channel ◽

Channel Trafficking ◽

Sorting Nexin ◽

Rate Limiting Step ◽

Surface Population ◽

Complex Protein ◽

Associated Proteins

The epithelial Na+ channel (ENaC) located at the apical membrane in many epithelia is the rate-limiting step for Na+ reabsorption. Tight regulation of the plasma membrane population of ENaC is required, as hypertension or hypotension may result if too many or too few ENaCs are present. Endocytosed ENaC travels to the early endosome and is then either trafficked to the lysosome for degradation or recycled back to the plasma membrane. Recently, the retromer recycling complex, located at the early endosome, has been implicated in plasma membrane protein recycling pathways. We hypothesized that the retromer is required for recycling of ENaC. Stabilization of retromer function with the retromer stabilizing chaperone R55 increased ENaC current, whereas knockdown or overexpression of individual retromer and associated proteins altered ENaC current and cell surface population of ENaC. KIBRA was identified as an ENaC-binding protein allowing ENaC to link to sorting nexin 4 to alter ENaC trafficking. Knockdown of the retromer-associated cargo-binding sorting nexin 27 protein did not alter ENaC current, whereas CCDC22, a CCC-complex protein, coimmunoprecipitated with ENaC, and CCDC22 knockdown decreased ENaC current and population at the cell surface. Together, our results confirm that retromer and the CCC complex play a role in recycling of ENaC to the plasma membrane.

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Sorting Nexin 27 (SNX27): A Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Trafficking

10.1101/304717 ◽

2018 ◽

Cited By ~ 1

Author(s):

Mark I. McDermott ◽

William R. Thelin ◽

Yun Chen ◽

Patrick T. Lyons ◽

Gabrielle Reilly ◽

...

Keyword(s):

Cystic Fibrosis ◽

Plasma Membrane ◽

Cell Surface ◽

Pdz Domain ◽

Type I ◽

Apical Surface ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Sorting Nexin ◽

Cystic Fibrosis Transmembrane

AbstractThe underlying defect in cystic fibrosis is mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel expressed at the apical surface of lung epithelia. In addition to its export and maintenance at the cell surface, CFTR regulation involves repeated cycles of transport through the endosomal trafficking system, including endocytosis and recycling. Many of the known disease mutations cause CFTR intracellular trafficking defects that result in failure of ion channel delivery to the apical plasma membrane. Corrective maneuvers directed at improving transport to the plasma membrane are thwarted by rapid internalization and degradation of the mutant CFTR proteins. The molecular mechanisms involved in these processes are not completely understood but may involve protein-protein interactions with the C-terminal type I PDZ-binding motif of CFTR. Using a proteomic approach, we identify sorting nexin 27 (SNX27) as a novel CFTR binding partner in human airway epithelial Calu-3 cells. SNX27 and CFTR interact directly, with the SNX27 PDZ domain being both necessary and sufficient for this interaction. SNX27 co-localizes with internalized CFTR at sub-apical endosomal sites in polarized Calu-3 cells, and either knockdown of the endogenous SNX27, or over-expression of a dominant-negative SNX27 mutant, resulted in significant decreases in cell surface CFTR levels. CFTR internalization was not affected by SNX27 knockdown, but defects were observed in the recycling arm of CFTR trafficking through the endosomal system. Furthermore, knockdown of SNX27 in Calu-3 cells resulted in significant decreases in CFTR protein levels, consistent with degradation of the internalized pool. These data identify SNX27 as a physiologically significant regulator of CFTR trafficking and homeostasis in epithelial cells.

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

The Journal of Cell Biology ◽

10.1083/jcb.200609161 ◽

2007 ◽

Vol 177 (1) ◽

pp. 115-125 ◽

Cited By ~ 108

Author(s):

Todd I. Strochlic ◽

Thanuja Gangi Setty ◽

Anand Sitaram ◽

Christopher G. Burd

Keyword(s):

Plasma Membrane ◽

Golgi Apparatus ◽

Rab Gtpase ◽

Endocytic Recycling ◽

Sorting Nexins ◽

Sorting Nexin ◽

Retromer Complex ◽

Iron Transporter ◽

Endocytic Sorting ◽

Recycling Pathway

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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Normal prion protein trafficking in cultured human erythroblasts

Blood ◽

10.1182/blood-2007-04-085183 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4518-4525 ◽

Cited By ~ 19

Author(s):

Rebecca E. Griffiths ◽

Kate J. Heesom ◽

David J. Anstee

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Prion Protein ◽

Protein Trafficking ◽

Prion Diseases ◽

Cross Linking ◽

Red Cell ◽

Jakob Disease ◽

Endosomal Pathway ◽

Tetraspanin Cd63

Normal prion protein (PrPc), an essential substrate for development of prion disease, is widely distributed in hematopoietic cells. Recent evidence that variant Creutzfeldt-Jakob disease can be transmitted by transfusion of red cell preparations has highlighted the need for a greater understanding of the biology of PrPc in blood and blood-forming tissues. Here, we show that in contrast to another glycosylphosphoinositol-anchored protein CD59, PrPc at the cell surface of cultured human erythroblasts is rapidly internalized through the endosomal pathway, where it colocalizes with the tetraspanin CD63. In the plasma membrane, PrPc colocalizes with the tetraspanin CD81. Cross-linking with anti-PrPc or anti-CD81 causes clustering of PrPc and CD81, suggesting they can share the same microdomain. These data are consistent with a role for tetraspanin-enriched microdomains in trafficking of PrPc. These results, when taken together with recent evidence that exosomes released from cells as a result of endosomal-mediated recycling to the plasma membrane contain prion infectivity, provide a pathway for the propagation of prion diseases.

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Sorting Nexin 10 as a Key Regulator of Membrane Trafficking in Bone-Resorbing Osteoclasts: Lessons Learned From Osteopetrosis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.671210 ◽

2021 ◽

Vol 9 ◽

Author(s):

Ari Elson ◽

Merle Stein ◽

Grace Rabie ◽

Maayan Barnea-Zohar ◽

Sabina Winograd-Katz ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Molecular Mechanisms ◽

Lessons Learned ◽

Model Systems ◽

Bone Homeostasis ◽

Sorting Nexin ◽

Sequence Of Events ◽

Ruffled Border ◽

Regulated Gene Expression

Bone homeostasis is a complex, multi-step process, which is based primarily on a tightly orchestrated interplay between bone formation and bone resorption that is executed by osteoblasts and osteoclasts (OCLs), respectively. The essential physiological balance between these cells is maintained and controlled at multiple levels, ranging from regulated gene expression to endocrine signals, yet the underlying cellular and molecular mechanisms are still poorly understood. One approach for deciphering the mechanisms that regulate bone homeostasis is the characterization of relevant pathological states in which this balance is disturbed. In this article we describe one such “error of nature,” namely the development of acute recessive osteopetrosis (ARO) in humans that is caused by mutations in sorting nexin 10 (SNX10) that affect OCL functioning. We hypothesize here that, by virtue of its specific roles in vesicular trafficking, SNX10 serves as a key selective regulator of the composition of diverse membrane compartments in OCLs, thereby affecting critical processes in the sequence of events that link the plasma membrane with formation of the ruffled border and with extracellular acidification. As a result, SNX10 determines multiple features of these cells either directly or, as in regulation of cell-cell fusion, indirectly. This hypothesis is further supported by the similarities between the cellular defects observed in OCLs form various models of ARO, induced by mutations in SNX10 and in other genes, which suggest that mutations in the known ARO-associated genes act by disrupting the same plasma membrane-to-ruffled border axis, albeit to different degrees. In this article, we describe the population genetics and spread of the original arginine-to-glutamine mutation at position 51 (R51Q) in SNX10 in the Palestinian community. We further review recent studies, conducted in animal and cellular model systems, that highlight the essential roles of SNX10 in critical membrane functions in OCLs, and discuss possible future research directions that are needed for challenging or substantiating our hypothesis.

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Mechanisms of Melanocortin-2 Receptor (MC2R) Internalization and Recycling in Human Embryonic Kidney (HEK) Cells: Identification of Key Ser/Thr (S/T) Amino Acids

Molecular Endocrinology ◽

10.1210/me.2011-0018 ◽

2011 ◽

Vol 25 (11) ◽

pp. 1961-1977 ◽

Cited By ~ 23

Author(s):

Simon Roy ◽

Sébastien Jean Roy ◽

Sandra Pinard ◽

Louis-Daniel Taillefer ◽

Mohamed Rached ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Molecular Mechanisms ◽

Surface Expression ◽

Receptor Internalization ◽

Cell Surface Expression ◽

Target Cells ◽

Camp Accumulation ◽

Human Embryonic Kidney ◽

And Function

Abstract ACTH is the most important stimulus of the adrenal cortex. The precise molecular mechanisms underlying the ACTH response are not yet clarified. The functional ACTH receptor includes melanocortin-2 receptor (MC2R) and MC2R accessory proteins (MRAP). In human embryonic kidney 293/Flp recombinase target cells expressing MC2R, MRAP1 isoforms, and MRAP2, we found that ACTH induced a concentration-dependent and arrestin-, clathrin-, and dynamin-dependent MC2R/MRAP1 internalization, followed by intracellular colocalization with Rab (Ras-like small guanosine triphosphate enzyme)4-, Rab5-, and Rab11-positive recycling endosomes. Preincubation of cells with monensin and brefeldin A revealed that 28% of the internalized receptors were recycled back to the plasma membrane and participated in total accumulation of cAMP. Moreover, certain intracellular Ser and Thr (S/T) residues of MC2R were found to play important roles not only in plasma membrane targeting and function but also in promoting receptor internalization. The S/T residues T131, S140, T204, and S280 were involved in MRAP1-independent cell-surface MC2R expression. Other mutants (S140A, S208A, and S202D) had lower cell-surface expressions in absence of MRAPβ. In addition, T143A and T147D drastically impaired cell-surface expression and function, whereas T131A, T131D, and S280D abrogated MC2R internalization. Thus, the modification of MC2R intracellular S/T residues may positively or negatively regulate its plasma membrane expression and the capacity of ACTH to induce cAMP accumulation. Mutations of T131, T143, T147, and S280 into either A or D had major repercussions on cell-surface expression, cAMP accumulation, and/or internalization parameters, pointing mostly to the second intracellular loop as being crucial for MC2R expression and functional regulation.

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K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway

The Journal of Cell Biology ◽

10.1083/jcb.200810114 ◽

2009 ◽

Vol 185 (3) ◽

pp. 493-502 ◽

Cited By ~ 157

Author(s):

Elsa Lauwers ◽

Christophe Jacob ◽

Bruno André

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Protein Trafficking ◽

Multivesicular Body ◽

Adapter Proteins ◽

Relative Importance ◽

Specific Signal ◽

Single Target ◽

Membrane Protein Trafficking ◽

Insight Into

A growing number of yeast and mammalian plasma membrane proteins are reported to be modified with K63-linked ubiquitin (Ub) chains. However, the relative importance of this modification versus monoubiquitylation in endocytosis, Golgi to endosome traffic, and sorting into the multivesicular body (MVB) pathway remains unclear. In this study, we show that K63-linked ubiquitylation of the Gap1 permease is essential for its entry into the MVB pathway. Carboxypeptidase S also requires modification with a K63-Ub chain for correct MVB sorting. In contrast, monoubiquitylation of a single target lysine of Gap1 is a sufficient signal for its internalization from the cell surface, and Golgi to endosome transport of the permease requires neither its ubiquitylation nor the Ub-binding GAT (Gga and Tom1) domain of Gga (Golgi localizing, gamma-ear containing, ARF binding) adapter proteins, the latter being crucial for subsequent MVB sorting of the permease. Our data reveal that K63-linked Ub chains act as a specific signal for MVB sorting, providing further insight into the Ub code of membrane protein trafficking.

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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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