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 basolateral plasma membrane domains of Madin-Darby canine kidney (MDCK) cells requires the N-ethylmaleimide-sensitive factor and is sensitive to botulinum neurotoxin serotype E (BoNT/E). BoNT/E is thought to selectively proteolyze the 25,000-dalton synaptosomal associated protein (SNAP-25), a protein found in neurons or cells of neuroendocrine origin. However, SNAP-25 is not found in MDCK cells. One possible target for BoNT/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 endocytic traffic of this protein. We find that: (i) SNAP-23 is expressed in MDCK cells and is found both at the basolateral plasma membrane and associated with apical and basolateral vesicles, (ii) canine SNAP-23 is cleaved by BoNT/E, (iii) transferrin recycling is N-ethylmaleimide-sensitive factor-dependent and BoNT/E-sensitive, and (iv) addition of either exogenous SNAP-23 or anti-SNAP-23 antibodies inhibits ligand recycling. Our observations suggest that SNAP-23 may be required for fusion of recycling vesicles with the basolateral membrane of polarized MDCK cells.
Leung
S M
SM
Laboratory of Epithelial Cell Biology, the Renal-Electrolyte Division of the Department of Medicine and Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.
Chen
D
D
DasGupta
B R
BR
Whiteheart
S W
SW
Apodaca
G
G
D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers; D004285:Dogs; D004705:Endocytosis; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; D013301:Streptolysins; D014168:Transferrin / Q000378:metabolism*
D002352:Carrier Proteins; D017931:DNA Primers; D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human; D013301:Streptolysins; D014168:Transferrin