Dissertation

Function and localization of Rab8 in the AP -1B mediated basolateral sorting pathway

Ang, Agnes Lee

2004

Overview

Polarized epithelial cells maintain plasma membrane asymmetry, which is vital to their function, by sorting apical and basolateral transmembrane proteins into different post-Golgi vesicles. Although it is well established that the small GTPase Rab8 is involved in the delivery of post-Golgi vesicles to the plasma membrane in various cell types (Moritz et al. 2001, Huber et al. 1993, 1995), its association with a specific trafficking pathway remained elusive. In addition there are at least two distinguishable pathways to the basolateral plasma membrane. One is taken by LDL receptor and VSVG proteins which contain tyrosine-based sorting signals. These basolateral proteins are sorted with the help of the epithelial specific clathrin-adaptor complex AP-1B. In contrast, another basolateral protein, Fc receptor, containing a dileucine-based sorting signal, is sorted independently of AP-1B expression (Fölsch et al 1999, 2001). Here we demonstrate a functional link between Rab8 and the AP-1B pathway. We found that rab8 localizes to a post-Golgi compartment and, in its activated form, causes the missorting of VSVG and LDLR from the basolateral to the apical surface as shown by confocal immunofluorescence microscopy and radioactive pulse-labeling studies. However, activated Rab8 had no effect on sorting of FcR. Furthermore we found that expression of activated Rab8 in LLC-PK1 kidney cells exogenously expressing AP-1B dramatically changes AP-1 localization. We hypothesize that Rab8 specifically regulates the AP-1B pathway perhaps by controlling AP-1B recruitment to post-Golgi membranes, specifically recycling endosomes (RE's). Localization of AP-1B to RE's along with other components (e.g., exocyst and Rab8) involved in AP-1B-dependent transport suggested that RE's may be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence that RE's play an essential role in basolateral transport in MDCK cells using four independent methods. Newly synthesized AP-1B dependent cargo, VSV-G, was found by video microscopy, immuno-EM, and cell fractionation to enter Tfn-positive RE's immediately upon exit from the TGN. Although transient, RE entry appears essential since enzymatic inactivation of RE's almost completely blocked VSV-G delivery to the cell surface. Since an apically targeted VSV G mutant behaves in a similar fashion, these results suggest that RE's may serve not only as an intermediate, but also as an important site for polarized sorting.

• Publisher: ProQuest Dissertations & Theses
• Subject: Cellular biology
• ISBN: 049672441X, 9780496724413