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Published 17 September 2001. doi:10.1083/jcb.200105138
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© The Rockefeller University Press, 0021-9525/2001/9/1197 $5.00
The Journal of Cell Biology, Volume 154, Number 6, September 17, 2001 1197-1208

Article

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells



Pamela L. Tuma1, Lydia K. Nyasae1, Jonathan M. Backer2 and Ann L. Hubbard1

1 Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD 21205
2 Department of Molecular Pharmacology, Albert Einstein College of Medicine, New York, NY 10461

Address correspondence to Ann L. Hubbard, Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, 725 N. Wolfe, Baltimore, MD 21205. Tel.: (410) 955-2337. Fax: (410) 955-1013. E-mail: alh{at}jhmi.edu

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089–1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI(3)P, was differentially regulating endocytosis from the two domains, we examined the PI(3)P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI(3)P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI(3)P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI(3)P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.

Key Words: transcytosis; endocytosis; hepatocytes; phosphoinositides; polarity


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