A bipartite signal regulates the faithful delivery of apical domain marker podocalyxin/Gp135.
Podocalyxin/Gp135 was recently demonstrated to participate in the formation of a preapical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation-rich region and the intracellular PDZ domain-binding motif. The function of this PDZ-binding motif could be substituted with a fusion construct of Gp135 with Ezrin-binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at the Golgi apparatus and facilitates oligomerization and sorting of Gp135 into a clustering complex. A defective connection between Gp135 and EBP50 or EBP50 knockdown results in a delayed exit from the detergent-resistant microdomain, failure of oligomerization, and basolateral missorting of Gp135. Furthermore, the basolaterally missorted EBP50-binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aid of its cytoplasmic partner EBP50.
Yu
Chun-Ying
CY
Department of Internal Medicine, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, 100 Taiwan.
Chen
Jen-Yau
JY
Lin
Yu-Yu
YY
Shen
Kuo-Fang
KF
Lin
Wei-Ling
WL
Chien
Chung-Liang
CL
ter Beest
Martin B A
MB
Jou
Tzuu-Shuh
TS
D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D016764:Cell Polarity; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis; D006031:Glycosylation; D021962:Membrane Microdomains / Q000378:metabolism; D008954:Models, Biological; D009154:Mutation; D011493:Protein Kinase C / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000235:genetics; D012795:Sialoglycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D000099149:Ezrin
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; D011493:Protein Kinase C; D021382:Protein Sorting Signals; D034741:RNA, Small Interfering; D012795:Sialoglycoproteins; D000099149:Ezrin; C001643:podocalyxin