Localization of scribble in  polarized hepatocytes

Laura M. Kallay, Lelita T. Braiterman, Pamela L. Tuma, Ya-Hui Chen and Ann L. Hubbard 

Scribble is a large, multi-domain protein important for the establishment and maintenance of epithelial cell polarity in D. melanogaster and C. elegans; however, its role in mammalian epithelial cells is largely unknown.  Scribble is a LAP (LRR (leucine rich repeats) and PDZ (PSD-95/Disc Large/ZO-1)) family member.  Family members share a structural organization consisting of sixteen amino-terminal LRRs followed by a LAP-specific domain of unknown function and either one or four carboxyl-terminal PDZ domains. We have examined the subcellular location of scribble in polarized hepatocytes using morphological, biochemical, and molecular approaches.  In WIF-B cells, scribble was found in multiple locations by indirect immunofluorescence: the basolateral plasma membrane, the tight junction region, in punctae at the cell-substrate, and within the cytoplasm.  Biochemically, scribble was greater than 90% membrane-associated.  Scribble was also present in purified rat liver plasma membrane sheets.  When the apical and basolateral domains were separated by sucrose density centrifugation, scribble distributed to a pelleted fraction, indicating its presence in a high density complex.  Scribble was insoluble in several different detergents further suggesting its presence in a membrane-associated multimeric complex.  

Figure 1

To map the domains that mediate its membrane localization, GFP-tagged scribble mutants were generated and expressed in WIF-B cells using recombinant adenoviruses.  The localizations of the GFP-tagged scribble EST, and four progressive truncation mutants, DLRR, DLRR+LAP and the PDZ1-4 + C-Term (the C-terminal half of scribble that containing the four PDZ domains), and  PDZ1-4 were similar to that of endogenous scribble: all five mutants were found at basolateral plasma membrane and in the tight junction region.  However, the construct encoding the C-terminal region beyond the PDZ domain was soluble. These show that in polarized hepatocytes the C-terminal half of scribble with four PDZ domains plays a role in its PM localization. Current experiments focus on: a) identifying the localization signal(s) in the C-terminal half of scribble; and b) characterizing the proposed plasma membrane complex and identifying interacting molecules.