title: Subcellular localisation of retromer in post-endocytic pathways of polarised Madin-Darby canine kidney cells.
abstract: BACKGROUND
  Retromer is required for endosome-to-Golgi retrieval of the cation-independent mannose 6-phosphate receptor (CI-MPR), allowing delivery of hydrolases into lysosomes. It is constituted by a conserved heterotrimer formed by vacuolar protein sorting (Vps) gene products Vps26, Vps35 and Vps29, which is in charge of cargo selection, and a dimer of phosphoinositide-binding sorting nexins (SNXs), which has a structural role. Retromer has been implicated in sorting of additional cargo. Thus, retromer also promotes polymeric immunoglobulin A (pIgA) transcytosis by the pIgA receptor (pIgR) in polarised cells, and considerable evidence implicates retromer in controlling epithelial cell polarity. However, the precise localisation of retromer along the endocytic pathway of polarised cells has not been studied in detail.
  
  
  RESULTS
  Our biochemical analysis using rat liver endosome fractions suggests a distinct distribution pattern. Although subunits of the cargo-selective complex were enriched in early endosomes (EEs), levels of SNX2 were greater in sorting endosomes. We then immunolocalised the retromer subunits in polarised Madin-Darby canine kidney (MDCK) cells by confocal microscopy. An estimated 25% of total Vps26 and SNX2 localised to EEs, with negligible portions in recycling endosomes as well as in late endosomes and lysosomes. Although Vps26 was in structures of more heterogeneous size and shape than SNX2, these markedly overlapped. In consequence, the two retromer subcomplexes mostly colocalised. When we analysed retromer overlap with its cargo, we found that structures retromer and pIgA(+) are independent of those structures retromer and CI-MPR(+) . Remarkably, retromer localised preferentially at the transcytotic pathway. Pharmacological inhibition of phosphoinositide 3-kinase affected the co-distribution of retromer with pIgA and the CI-MPR, delaying pIgA progress to the apical surface.
  
  
  CONCLUSIONS
  In polarised MDCK cells, we found retromer associated with certain specialised EE-derived pathways. Our data imply that retromer is largely engaged in pIgA transcytosis in pIgR-expressing MDCK cells, as opposed to endosome-to-Golgi retrieval.
authors.affiliation: Laboratory of Epithelial Cell Biology, Príncipe Felipe Research Center (CIPF), Valencia, 46012, Spain.
authors.forename: Maravillas
authors.initials: M
authors.lastname: Mellado
authors.forename: Yasmina
authors.initials: Y
authors.lastname: Cuartero
authors.forename: Ramon
authors.initials: R
authors.lastname: Brugada
authors.forename: Marcel
authors.initials: M
authors.lastname: Verges
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D016764:Cell Polarity / Q000502:physiology*; D004285:Dogs; D011992:Endosomes / Q000378:metabolism / Q000502:physiology*; D006056:Golgi Apparatus / Q000378:metabolism / Q000502:physiology; D006801:Humans; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D058305:Sorting Nexins / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D058305:Sorting Nexins; D033921:Vesicular Transport Proteins; D019869:Phosphatidylinositol 3-Kinases
keywords: Polarised trafficking; Retromer; Sorting nexin; Transcytosis