SNAP-23 REQUIREMENT FOR TRANSFERRIN RECYCLING IN STREPTOLYSIN-O-PERMEABILIZED MADIN-DARBY CANINE KIDNEY-CELLS

Fusion of recycling and transcytotic vesicles with the apical and baso lateral plasma membrane domains of Madin-Darby canine kidney (MDCK) ce lls requires the N-ethylmaleimide-sensitive factor and is sensitive to botulinum neurotoxin serotype E (BoNT/E). BoNT/E is thought to select ively proteolyze the 25,000-dalton synaptosomal associated protein (SN AP-25), a protein found in neurons or cells of neuroendocrine origin. However, SNAP-25 is not found in MDCK cells. One possible target for B oNT/E in MDCK cells is SNAP-23, a newly described SNAP-25 homolog that is found in several organs including kidney. Currently, the function of SNAP-23 is unknown. We have reconstituted transferrin recycling in permeabilized MDCK cells to assess the role of SNAP-23 in the endocyti c traffic of this protein. We find that: (i) SNAP-23 is expressed in M DCK cells and is found both at the basolateral plasma membrane and ass ociated with apical and basolateral vesicles, (ii) canine SNAP-23 is c leaved by BoNT/E, (iii) transferrin recycling is N-ethylmaleimide-sens itive factor-dependent and BoNT/E-sensitive, and (iv) addition of eith er exogenous SNAP-23 or anti-SNAP-23 antibodies inhibits ligand recycl ing. Our observations suggest that SNAP-23 may be required for fusion of recycling vesicles with the basolateral membrane of polarized MDCK cells.