Molecular and Cellular Biochemistry

, Volume 366, Issue 1–2, pp 159–168 | Cite as

SNARE proteins are not excessive for the formation of post-Golgi SNARE complexes in HeLa cells

  • Miki Okayama
  • Akiko Shitara
  • Toshiya Arakawa
  • Yoshifumi Tajima
  • Itaru Mizoguchi
  • Taishin Takuma
Article
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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.

Keywords

Exocytosis Constitutive secretion siRNA SNARE SNARE complex 

Abbreviations

SNARE

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor

GFP

Green fluorescence protein

VAMP

Vesicle-associated membrane protein

hGH–GFP

Human growth hormone tagged with GFP

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Notes

Acknowledgments

This study was supported in part by a grant-in-aid for scientific research (No.19592153) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Supplementary material

11010_2012_1293_MOESM1_ESM.jpg (518 kb)
Fig. S1 Effects of gene silencing of VAMP3, 5, 7, and 8 on the levels of corresponding proteins and mRNAs in HeLa cells. HeLa cells were treated twice with 100 nM siRNA for each VAMP or with 100 nM control siRNA. The levels of mRNA and protein were determined at 48 and 72 h, respectively, after the first introduction of siRNAs. a The levels of VAMP3, VAMP8, and β-actin in the cell lysates were analyzed by immunoblotting. The mRNA levels for VAMP5 (b), VAMP7 (c), and GAPDH were estimated by real-time PCR with CYBR green. (JPEG 517 kb)
11010_2012_1293_MOESM2_ESM.jpg (438 kb)
Fig. S2 Effects of gene silencing of VAMP3, 5, 7, and 8 on the cell/medium ratio of CLuc activity. HeLa cells, stably expressing CLuc, were treated twice with 100 nM siRNAs against each VAMP or with 100 nM control siRNA. The CLuc activities in incubation medium and cells were determined 72 h after the first introduction of siRNA. a CLuc activities in the incubation medium. b CLuc activities in the cell lysates. c Cell/medium ratios (×100) of CLuc activity. Data shown are the means ± SD (n = 4) of a typical experiment from three separate experiments. (JPEG 437 kb)
11010_2012_1293_MOESM3_ESM.jpg (982 kb)
Fig. S3 Effects of gene silencing of VAMP3, 5, 7, and 8 on the constitutive exocytotic pathway of hGH–GFP. HeLa cells, which stably expressed hGH–GFP, were treated twice with 100 nM siRNA against each syntaxin or with 100 nM control siRNA. Two days after the first introduction of siRNAs, 2 mM Na-butyrate was added to the medium for the stimulation of hGH–GFP synthesis in the cells; and 24 h after the stimulation, the cells were fixed with 4 % paraformaldehyde in PBS for 15 min. Scale bars = 10 m. (JPEG 981 kb)

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Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  • Miki Okayama
    • 1
  • Akiko Shitara
    • 2
  • Toshiya Arakawa
    • 2
  • Yoshifumi Tajima
    • 3
  • Itaru Mizoguchi
    • 1
  • Taishin Takuma
    • 2
  1. 1.Department of Orthodontics, School of DentistryHealth Sciences University of HokkaidoTobetsuJapan
  2. 2.Department of Biochemistry, School of DentistryHealth Sciences University of HokkaidoTobetsuJapan
  3. 3.Department of Oral Pathology, School of DentistryMeikai UniversitySakadoJapan

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