Dynamic analysis identifies novel roles for DLG-1 subdomains in AJM-1 recruitment and LET-413-dependent apical focusing.
Cell-cell junctions are composed of a diverse array of specialized proteins that are necessary for the movement and integrity of epithelia. Scaffolding molecules, such as membrane-associated guanylate kinases (MAGUKs) contain multiple protein-protein interaction domains that integrate these proteins into macromolecular complexes at junctions. We have used structure-function experiments to dissect the role of domains of the Caenorhabditis elegans MAGUK DLG-1, a homolog of Drosophila Discs large and vertebrate SAP97. DLG-1 deletion constructs were analyzed in directed yeast two-hybrid tests as well as in vivo in a dlg-1 null mutant background. Our studies identify novel roles for several key domains. First, the L27 domain of DLG-1 mediates the physical interaction of DLG-1 with its binding partner, AJM-1, as well as DLG-1 multimerization. Second, the PDZ domains of DLG-1 mediate its association with the junction. Third, using dynamic in vivo imaging, we demonstrate that the SH3 domain is required for rapid lateral distribution of DLG-1 via a LET-413/Scribble-dependent pathway. Finally, we found that inclusion of the SH3 domain can ameliorate dlg-1 mutant phenotypes, but full rescue of lethality required the complete C terminus, which includes the GUK and Hook domains, thereby demonstrating the importance of the C-terminus for DLG-1 function. Our results represent the first in vivo analysis of requirements for the L27 domain of a Discs-large/SAP97 protein, identify a crucial LET-413/Scribble regulatory motif and provide insight into how MAGUK subdomains function to maintain epithelial integrity during development.
Lockwood
Christopher A
CA
Program in Genetics, University of Wisconsin-Madison, Madison, WI 53706, USA.
Lynch
Allison M
AM
Hardin
Jeff
J
D000483:Alleles; D000818:Animals; D017173:Caenorhabditis elegans / Q000166:cytology* / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D051528:Guanylate Kinases / Q000737:chemistry / Q000378:metabolism*; D007365:Intercellular Junctions / Q000378:metabolism; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D013329:Structure-Activity Relationship
C441580:AJM-1 protein, C elegans; D029742:Caenorhabditis elegans Proteins; C413362:LET-413 protein, C elegans; D050505:Mutant Proteins; C530790:DLG-1 protein, C elegans; D051528:Guanylate Kinases