Maria Bastaki

Past Department Affiliation:1998-2001, Post-Doctoral Fellow in Cell Biology 

Present Department Affiliation: Environmental Health Sciences

                                                           School of Public health

                                                           219 Warren Hall

                                                           University of California at Berkeley

                                                           Berkeley , CA 94720-7360

Education: B.Sc. in Pharmacy from the University of Patras, Greece; Ph.D. in Pharmacology from the University of Patras Medical School, Greece. 

Telephone Number: 510-643 5349

Fax Number: 510-642 0427

Email Address: mbastaki@uclink.berkeley.edu

 

   

Absence of a Direct Apical Transport Pathway for a Single Transmembrane Domain Protein in Hepatocytes

In polarized MDCK cells, the newly synthesized apical plasma membrane (PM) protein dipeptidylpeptidase IV (DPPIV) can be delivered to the apical PM domain either directly from the TGN, or indirectly via transcytosis from the basolateral domain (Casanova etal, 1991, J.Biol.Chem., 266(36): 24428-24432). However, a mutant soluble form of DPPIV (sDPPIV), generated by cleavage of the transmembrane domain in the ER, follows only the direct route, suggesting that the extracellular domain of DPPIV contains positive apical targeting information (Weisz etal, 1992, J.Biol.Chem., 267(31): 22282-22288). Here, we used sDPPIV as a reporter protein to determine whether a direct route of apical delivery operates in hepatocytes. We used recombinant adenovirus technology to express sDPPIV in vitro in the polarized hepatic cell line WIF-B and in vivo. In WIF-B cells, sDPPIV was detected only in the culture medium (basolateral secretion), and not in the bile canalicular (apical) space. Similarly in vivo, sDPPIV was overwhelmingly present in the plasma (basolateral secretion). Metabolic labeling showed that the amount of 35S-sDPPIV found in bile was comparable to that of 35S-albumin, a basolaterally secreted plasma protein, which reaches bile via transcytosis from the circulation. Our results suggest that, in hepatocytes, there is no direct apical delivery mechanism for single transmembrane domain proteins, such as DPPIV.

Indirect transport of apical single transmembrane domain (TMD) proteins in hepatocytes.

We have previously shown that the apical single TMD protein, DPPIV, is transported to the apical plasma membrane (PM) of hepatic cells only by the indirect route, via the basolateral PM (Bastaki et.al. 1999, Mol. Biol. Cell, 10(Sup): 311a). This is in contrast to simple epithelia, where single TMD proteins are targeted directly from the TGN to the appropriate PM domain. We have extended our studies using two other single TMD proteins, the polymeric immunoglobulin receptor (pIgR) and the influenza virus hemagglutinin (HA). We expressed the wild type (wt) and truncated secreted (s) forms of these two proteins in WIF-B cells in vitro and in rat liver in vivo. The secreted forms, s-pIgR and sHA contain putative apical targeting signals. In MDCK cells, they are directly transported from the TGN to the apical PM. HA in particular is a model protein of direct apical transport. However, in WIF-B cells, wt-HA followed the indirect (transcytotic) route. Consistently, the s-pIgR and sHA were secreted from the basolateral surface of WIF-B cells into the medium, indicating that their route of transport is also indirect in hepatic cells. No secretion into the apical (bile canalicular) space was detected. We obtained similar results in vivo, under conditions where liver tissue is the main site of viral infection and reproducibly accounts for >95% of the expressed protein. Newly synthesized s-pIgR and sHA were secreted into the plasma, with very small amounts detected in the bile, and at levels comparable to that of basolaterally-secreted albumin. Thus, our data support the hypothesis that there is no direct delivery mechanism for apical single TMD proteins in hepatic cells (NIH, P01 DK44375).