title: SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells.
abstract: To evaluate the role of SNARE proteins in the constitutive exocytosis, we knocked down syntaxin 3, 4, 5, 6, 7, and VAMP3, 5, 7, 8 with their siRNAs, and determined the cell-to-medium ratio of CLuc, a secreted luciferase of Cypridina noctiluca. Although the protein level of SNAREs in HeLa cells was markedly reduced by the siRNA treatment, the cell/medium ratio was scarcely increased by any siRNAs except for syntaxin 5. The accumulation of GFP-tagged human growth hormone was also visible only by the knockdown of syntaxin 5. To examine whether the residual amount of SNAREs are sufficient for maintaining normal constitutive exocytosis, we estimated the effect of siRNAs on the level of post-Golgi SNARE complexes containing syntaxin 4, SNAP23, and VAMP3 or VAMP8. The amount of SNARE complexes was robustly decreased by siRNAs and was well correlated with the residual amount of SNAREs in the lysates, suggesting that SNAREs are unnecessarily excessive for the formation of post-Golgi SNARE complexes in HeLa cells.
authors.affiliation: Department of Orthodontics, School of Dentistry, Health Sciences University of Hokkaido, Tobetsu, Hokkaido 061-0293, Japan.
authors.forename: Miki
authors.initials: M
authors.lastname: Okayama
authors.forename: Akiko
authors.initials: A
authors.lastname: Shitara
authors.forename: Toshiya
authors.initials: T
authors.lastname: Arakawa
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Tajima
authors.forename: Itaru
authors.initials: I
authors.lastname: Mizoguchi
authors.forename: Taishin
authors.initials: T
authors.lastname: Takuma
mesh_terms: D015870:Gene Expression; D055785:Gene Knockdown Techniques; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000378:metabolism; D006367:HeLa Cells; D019382:Human Growth Hormone / Q000096:biosynthesis / Q000378:metabolism; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000378:metabolism; D008561:Membrane Fusion; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism; D050765:Qa-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D050683:R-SNARE Proteins / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D055571:Secretory Pathway
chemical_list: D046912:Multiprotein Complexes; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D008156:Luciferases