The endosomal/lysosomal transmembrane protein CLN3 is mutated in the Batten disease (juvenile neuronal ceroid lipofuscinosis, JNCL). However, the molecular mechanism of JNCL pathogenesis and the exact function of the CLN3 protein have remained unclear. Previous studies have shown that deletion of BTN1, the yeast orthologue of CLN3, leads to increased expression of BTN2. BTN2 encodes Btn2p, a proposed homologue to a novel microtubule-binding protein Hook1, which regulates endocytosis in Drosophila. We analysed here the putative interconnection between CLN3 and Hook1 in the mammalian cells and discovered that overexpression of human CLN3 induces aggregation of Hook1 protein, potentially by mediating its dissociation from the microtubules. Using in vitro binding assay we were able to demonstrate a weak interaction between Hook1 and the cytoplasmic segments of CLN3. We also found receptor-mediated endocytosis to be defective in CLN3-deficient JNCL fibroblasts, connecting CLN3, Hook1 and endocytosis in the mammalian system. Moreover, co-immunoprecipitation experiments showed that Hook1 physically interacts with endocytic Rab7, Rab9 and Rab11, hence delineating a manifold role for mammalian Hook1 in membrane trafficking events. These novel interactions between the microtubule-binding Hook1 and the large family of Rab GTPases also suggest a link between CLN3 function, microtubule cytoskeleton and endocytic membrane trafficking.

Background: BACH1 (BRCA1-associated C-terminal helicase 1; also known as BRCA1-interacting protein 1, BRIP1) is a helicase protein that interacts in vivo with BRCA1, the protein product of one of the major genes for hereditary predisposition to breast cancer. Previously, two BACH1 germ line missense mutations have been identified in early-onset breast cancer patients with and without family history of breast and ovarian cancer.

CLN3 is a transmembrane protein with a predominant localization in lysosomes in non-neuronal cells but isIn the present study, we investigated the pathways and mechanisms of CLN3 sorting using biochemical binding assays and immunofluorescence methods. The dileucine motif of CLN3 bound both AP-1 and AP-3 in vitro, and expression of mutated CLN3 in AP-1-or AP-3-deficient mouse fibroblasts showed that both adaptor complexes are required for sequential sorting of CLN3 via this motif. Our data indicate the involvement of complex sorting machinery in the trafficking of CLN3 and emphasize the diversity of parallel and sequential sorting pathways in the trafficking of membrane proteins.