Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells.
In pancreatic beta-cells, the syntaxin 6 (Syn6) soluble N-ethylmaleimide-sensitive factor attachment protein receptor is distributed in the trans-Golgi network (TGN) (with spillover into immature secretory granules) and endosomes. A possible Syn6 requirement has been suggested in secretory granule biogenesis, but the role of Syn6 in live regulated secretory cells remains unexplored. We have created an ecdysone-inducible gene expression system in the INS-1 beta-cell line and find that induced expression of a membrane-anchorless, cytosolic Syn6 (called Syn6t), but not full-length Syn6, causes a prominent defect in endosomal delivery to lysosomes, and the TGN, in these cells. The defect occurs downstream of the endosomal branchpoint involved in transferrin recycling, and upstream of the steady-state distribution of mannose 6-phosphate receptors. By contrast, neither acquisition of stimulus competence nor the ultimate size of beta-granules is affected. Biosynthetic effects of dominant-interfering Syn6 seem limited to slowed intragranular processing to insulin (achieving normal levels within 2 h) and minor perturbation of sorting of newly synthesized lysosomal proenzymes. We conclude that expression of the Syn6t mutant slows a rate-limiting step in endosomal maturation but provides only modest and potentially indirect interference with regulated and constitutive secretory pathways, and in TGN sorting of lysosomal enzymes.
Kuliawat
Regina
R
Division of Endocrinology and Department of Developmental/Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Kalinina
Elena
E
Bock
Jason
J
Fricker
Lloyd
L
McGraw
Timothy E
TE
Kim
Se Ryoung
SR
Zhong
Jiayu
J
Scheller
Richard
R
Arvan
Peter
P
D000418:Albumins / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002401:Cathepsin B / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005089:Exocytosis; D005799:Genes, Dominant; D007515:Islets of Langerhans / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D009154:Mutation; D011233:Precipitin Tests; D050765:Qa-SNARE Proteins; D051381:Rats; D050600:SNARE Proteins; D022142:Secretory Vesicles / Q000378:metabolism; D012672:Semliki forest virus / Q000378:metabolism; D013395:Sucrose / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000502:physiology*; D021601:trans-Golgi Network / Q000378:metabolism*
D000418:Albumins; D008565:Membrane Proteins; D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D014168:Transferrin; D033921:Vesicular Transport Proteins; D013395:Sucrose; D004247:DNA; D002401:Cathepsin B