Intracellular location of aquaporin-2 serine 269 phosphorylation and dephosphorylation in kidney collecting duct cells

2020 ◽  
Vol 319 (4) ◽  
pp. F592-F602
Author(s):  
Kit Yee Wong ◽  
Wei-Ling Wang ◽  
Shih-Han Su ◽  
Chin-Fu Liu ◽  
Ming-Jiun Yu
Keyword(s):  
Collecting Duct ◽  
Protein Sorting ◽  
Apical Plasma Membrane ◽  
Water Reabsorption ◽  
Intracellular Location ◽  
Vacuolar Protein Sorting ◽  
Recycling Endosomes ◽  
Aquaporin 2 ◽  
Early Endosomes ◽  
Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for water reabsorption by the kidney collecting ducts. Under control conditions, most AQP2 resides in the recycling endosomes of principal cells, where it answers to vasopressin with trafficking to the apical plasma membrane to increase water reabsorption. Upon vasopressin withdrawal, apical AQP2 retreats to the early endosomes before joining the recycling endosomes for the next vasopressin stimulation. Prior studies have demonstrated a role of AQP2 S269 phosphorylation in reducing AQP2 endocytosis, thereby prolonging apical AQP2 retention. Here, we studied where in the cells S269 was phosphorylated and dephosphorylated in response to vasopressin versus withdrawal. In mpkCCD collecting cells, vacuolar protein sorting 35 knockdown slowed vasopressin-induced apical AQP2 trafficking, resulting in AQP2 accumulation in the recycling endosomes where S269 was phosphorylated. Rab7 knockdown, which impaired AQP2 trafficking from the early to recycling endosomes, reduced vasopressin-induced S269 phosphorylation. Rab5 knockdown, which impaired AQP2 endocytosis, did not affect vasopressin-induced S269 phosphorylation. Upon vasopressin withdrawal, S269 was not dephosphorylated in Rab5 knockdown cells. In contrast, S269 dephosphorylation upon vasopressin withdrawal was completed in Rab7 or vacuolar protein sorting 35 knockdown cells. We conclude that S269 is dephosphorylated during Rab5-mediated AQP2 endocytosis before AQP2 joins the recycling endosomes upon vasopressin withdrawal. While in the recycling endosomes, AQP2 can be phosphorylated at S269 in response to vasopressin before apical trafficking.

scholarly journals Rab7 involves Vps35 to mediate AQP2 sorting and apical trafficking in collecting duct cells

2020 ◽  
Vol 318 (4) ◽  
pp. F956-F970 ◽  
Author(s):  
Wei-Ling Wang ◽  
Shih-Han Su ◽  
Kit Yee Wong ◽  
Chan-Wei Yang ◽  
Chin-Fu Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Water Reabsorption ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Vacuolar Protein

Aquaporin-2 (AQP2) is a vasopressin-regulated water channel protein responsible for osmotic water reabsorption by kidney collecting ducts. In response to vasopressin, AQP2 traffics from intracellular vesicles to the apical plasma membrane of collecting duct principal cells, where it increases water permeability and, hence, water reabsorption. Despite continuing efforts, gaps remain in our knowledge of vasopressin-regulated AQP2 trafficking. Here, we studied the functions of two retromer complex proteins, small GTPase Rab7 and vacuolar protein sorting 35 (Vps35), in vasopressin-induced AQP2 trafficking in a collecting duct cell model (mpkCCD cells). We showed that upon vasopressin removal, apical AQP2 returned to Rab5-positive early endosomes before joining Rab11-positive recycling endosomes. In response to vasopressin, Rab11-associated AQP2 trafficked to the apical plasma membrane before Rab5-associated AQP2 did so. Rab7 knockdown resulted in AQP2 accumulation in early endosomes and impaired vasopressin-induced apical AQP2 trafficking. In response to vasopressin, Rab7 transiently colocalized with Rab5, indicative of a role of Rab7 in AQP2 sorting in early endosomes before trafficking to the apical membrane. Rab7-mediated apical AQP2 trafficking in response to vasopressin required GTPase activity. When Vps35 was knocked down, AQP2 accumulated in recycling endosomes under vehicle conditions and did not traffic to the apical plasma membrane in response to vasopressin. We conclude that Rab7 and Vps35 participate in AQP2 sorting in early endosomes under vehicle conditions and apical membrane trafficking in response to vasopressin.

scholarly journals Depletion of vacuolar protein sorting-associated protein 35 is associated with increased lysosomal degradation of aquaporin-2

2016 ◽  
Vol 311 (6) ◽  
pp. F1294-F1307 ◽  
Author(s):  
Mi Suk Lee ◽  
Hyo-Jung Choi ◽  
Eui-Jung Park ◽  
Hye-Jeong Park ◽  
Tae-Hwan Kwon
Keyword(s):  
Posttranslational Modifications ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Protein Sorting ◽  
Protein Abundance ◽  
Lysosomal Degradation ◽  
Vacuolar Protein Sorting ◽  
Aquaporin 2 ◽  
Cell Surface Biotinylation ◽  
Vacuolar Protein

The carboxyl terminus of aquaporin-2 (AQP2c) undergoes posttranslational modifications, including phosphorylation and ubiquitination, in the process of regulating aquaporin-2 (AQP2) translocation and protein abundance. We aimed to identify novel proteins interacting with AQP2c. Recombinant AQP2c protein was made in Escherichia coli BL21 (DE3) cells by exploiting the pET32 TrxA fusion system. Lysates of rat kidney inner medullary collecting duct (IMCD) tubule suspensions interacted with rat AQP2c bound to Ni2+-resin were subjected to LC-MS/MS proteomic analysis. Potential interacting proteins were identified, including vacuolar protein sorting-associated protein 35 (Vps35). Coimmunoprecipitation assay demonstrated that Vps35 interacted with AQP2c. Immunohistochemistry of rat kidney revealed that AQP2 and Vps35 were partly colocalized at the intracellular vesicles in collecting duct cells. The role of Vps35 in AQP2 regulation induced by 1-deamino-8D-arginine vasopressin (dDAVP) was examined in mpkCCDc14 cells. Cell surface biotinylation assay demonstrated that dDAVP-induced apical translocation of AQP2 was significantly decreased under siRNA-mediated Vps35 knockdown. dDAVP-induced AQP2 upregulation was less prominent in the cells with Vps35 knockdown. Moreover, AQP2 protein abundance was decreased to a greater extent during the withdrawal period after dDAVP stimulation under Vps35 knockdown, which was significantly inhibited by chloroquine (a blocker of the lysosomal pathway) but not by MG132 (a proteasome inhibitor). Immunocytochemistry demonstrated that internalized AQP2 was more associated with lysosomal-associated membrane protein 1 (LAMP-1) in primary cultured IMCD cells under a Vps35 knockdown situation. Taken together, our results show that Vps35 interacts with AQP2c, and depletion of Vps35 is likely to be associated with decreased AQP2 trafficking and increased lysosomal degradation of AQP2 protein.

Advances in aquaporin-2 trafficking mechanisms and their implications for treatment of water balance disorders

2020 ◽  
Vol 319 (1) ◽  
pp. C1-C10 ◽  
Author(s):  
Robert A. Fenton ◽  
Sathish K. Murali ◽  
Hanne B. Moeller
Keyword(s):  
Protein Interactions ◽  
Collecting Duct ◽  
Water Channel ◽  
Plasma Osmolality ◽  
Carboxyl Terminus ◽  
Apical Plasma Membrane ◽  
Tail Domain ◽  
Water Reabsorption ◽  
Aquaporin 2

In mammals, conservation of body water is critical for survival and is dependent on the kidneys’ ability to minimize water loss in the urine during periods of water deprivation. The collecting duct water channel aquaporin-2 (AQP2) plays an essential role in this homeostatic response by facilitating water reabsorption along osmotic gradients. The ability to increase the levels of AQP2 in the apical plasma membrane following an increase in plasma osmolality is a rate-limiting step in water reabsorption, a process that is tightly regulated by the antidiuretic hormone arginine vasopressin (AVP). In this review, the focus is on the role of the carboxyl-terminus of AQP2 as a key regulatory point for AQP2 trafficking. We provide an overview of AQP2 structure, disease-causing mutations in the AQP2 carboxyl-terminus, the role of posttranslational modifications such as phosphorylation and ubiquitylation in the tail domain, and their implications for balanced trafficking of AQP2. Finally, we discuss how various modifications of the AQP2 tail facilitate selective protein-protein interactions that modulate the AQP2 trafficking mechanism.

scholarly journals Vps4-A (vacuolar protein sorting 4-A) is a binding partner for a novel Rho family GTPase, Rnd2

2002 ◽  
Vol 365 (2) ◽  
pp. 349-353 ◽  
Author(s):  
Hiroko TANAKA ◽  
Hirotada FUJITA ◽  
Hironori KATOH ◽  
Kazutoshi MORI ◽  
Manabu NEGISHI
Keyword(s):  
Protein Sorting ◽  
Yeast Two Hybrid ◽  
Vacuolar Protein Sorting ◽  
Aaa Atpase ◽  
Early Endosomes ◽  
Rho Family Gtpases ◽  
Rho Family ◽  
Vacuolar Protein ◽  
Two Hybrid ◽  
Vacuolar Protein Sorting 4

Rho family GTPases are implicated in a variety of biological activities, including endocytic vesicle trafficking. Rnd2 is a new member of Rho family GTPases, but its biological functions are not known. In the present study, we have performed a yeast two-hybrid screening using Rnd2 as bait and revealed that Rnd2 binds specifically to Vps4-A (where Vsp4-A is vacuolar protein sorting 4-A), a member of the AAA ATPase family and a central regulator for early endosome trafficking. This interaction was determined by the yeast two-hybrid system, in vitro binding and co-immunoprecipitation studies. Vps4-A associated with both guanosine 5′-[β-thio]triphosphate-bound active and guanosine 5′-[β-thio]diphosphate-bound inactive forms of Rnd2. An ATPase-defective Vps4-A mutant, Vps4-AE228Q, expressed in HeLa cells was accumulated in the early endosomes. When Rnd2 was co-expressed with Vps4-AE228Q, Rnd2 was recruited to the Vps4-A-bound early endosomes. These results suggest that Rnd2 is involved in the regulation of endosomal trafficking via direct binding to Vps4-A.

scholarly journals An evolutionary switch in the specificity of an endosomal CORVET tether underlies formation of regulated secretory vesicles in the ciliate Tetrahymena thermophila

2017 ◽  
Author(s):  
Daniela Sparvoli ◽  
Elisabeth Richardson ◽  
Hiroko Osakada ◽  
Xun Lan ◽  
Masaaki Iwamoto ◽  
...  
Keyword(s):  
Tetrahymena Thermophila ◽  
Secretory Granules ◽  
Protein Sorting ◽  
Endocytic Pathway ◽  
Forward Genetics ◽  
Vacuolar Protein Sorting ◽  
Protein Receptor ◽  
Early Endosomes ◽  
Class C ◽  
Vacuolar Protein

SummaryIn the endocytic pathway of animals, two related complexes, called CORVET (Class C Core Vacuole/Endosome Transport) and HOPS (Homotypic fusion and protein sorting), act as both tethers and fusion factors for early and late endosomes, respectively. Mutations in CORVET or HOPS lead to trafficking defects and contribute to human disease including immune dysfunction. HOPS and CORVET are conserved throughout eukaryotes but remarkably, in the ciliate Tetrahymena thermophila, the HOPS-specific subunits are absent while CORVET-specific subunits have proliferated. VPS8 (Vacuolar Protein Sorting), a CORVET subunit, expanded to 6 paralogs in Tetrahymena. This expansion correlated with loss of HOPS within a ciliate subgroup including the Oligohymenophorea, which contains Tetrahymena. As uncovered via forward genetics, a single VPS8 paralog in Tetrahymena (VPS8A) is required to synthesize prominent secretory granules called mucocysts. More specifically, ∆vps8a cells fail to deliver a subset of cargo proteins to developing mucocysts, instead accumulating that cargo in vesicles also bearing the mucocyst sorting receptor, Sor4p. Surprisingly, although this transport step relies on CORVET, it does not appear to involve early endosomes. Instead, Vps8a associates with the late endosomal/lysosomal marker Rab7, indicating target specificity switching occurred in CORVET subunits during the evolution of ciliates. Mucocysts belong to a markedly diverse and understudied class of protist secretory organelles called extrusomes. Our results underscore that biogenesis of mucocysts depends on endolysosomal trafficking, revealing parallels with invasive organelles in apicomplexan parasites and suggesting that a wide array of secretory adaptations in protists, like in animals, depend on mechanisms related to lysosome biogenesis.AbbreviationsLRO(Lysosome-related organelle)HOPS(homotypic fusion and protein sorting complex)CORVET(Class C core Vacuole/Endosome Transport)VPS(vacuolar protein sorting)GRL(granule lattice)GRT(granule tip)Igr(Induced upon granule regeneration)SNARE(Soluble NSF attachment protein receptor)LECA(last eukaryotic common ancestor)

scholarly journals VPS21Controls Entry of Endocytosed and Biosynthetic Proteins into the Yeast Prevacuolar Compartment

2000 ◽  
Vol 11 (2) ◽  
pp. 613-626 ◽  
Author(s):  
Sonja R. Gerrard ◽  
Nia J. Bryant ◽  
Tom H. Stevens
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Membrane Fusion ◽  
Protein Sorting ◽  
Vacuolar Protein Sorting ◽  
Endosomal Compartment ◽  
Early Endosomes ◽  
Strong Homology ◽  
Rab Family ◽  
Prevacuolar Compartment ◽  
Vacuolar Protein

Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class DVPS genes include VPS21,PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.

scholarly journals An Interorganellar Bidding War: Vps13 Localization by Competitive Organelle-Specific Adaptors

Contact ◽  
2018 ◽  
Vol 1 ◽  
pp. 251525641881462
Author(s):  
Samantha K. Dziurdzik ◽  
Björn D.M. Bean ◽  
Elizabeth Conibear
Keyword(s):  
Endoplasmic Reticulum ◽  
Recent Work ◽  
Protein Sorting ◽  
Repeat Region ◽  
Vacuolar Protein Sorting ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Prospore Membrane ◽  
Membrane Contact ◽  
Vacuolar Protein

Membrane contact sites are regulated through the controlled recruitment of constituent proteins. Yeast vacuolar protein sorting 13 (Vps13) dynamically localizes to membrane contact sites at endosomes, vacuoles, mitochondria, and the endoplasmic reticulum under different cellular conditions and is recruited to the prospore membrane during meiosis. Prior to our recent work, the mechanism for localization at contact sites was largely unknown. We identified Ypt35 as a novel Vps13 adaptor for endosomes and the nucleus-vacuole junction. Furthermore, we discovered a conserved recruitment motif in Ypt35 and found related motifs in the prospore membrane and mitochondrial adaptors, Spo71 and Mcp1, respectively. All three adaptors compete for binding to a six-repeat region of Vps13, suggesting adaptor competition regulates Vps13 localization. Here, we summarize and discuss the implications of our work, highlighting key outstanding questions.

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