R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed

Nandhakumar Thayanidhi, Yingjian Liang, Haruki Hasegawa, Deborah C. Nycz, Viola Oorschot, Judith Klumperman, Jesse C. Hay

Research output: Contribution to journalArticlepeer-review

Abstract

Background information: The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. Results: A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. Conclusions: The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology. Ykt6 is an unconventional R-SNARE protein that resides in membrane-associated protease-resistant protein particles. Over-expression of ykt6 in cultured cells alters cell cycle progression. Surprisingly, neither the formation of protein particles nor the cell cycle effects require the C-terminal lipidation of ykt6 thought to be critical for SNARE function. The particulate structures and cell cycle effects may arise from novel features of the ykt6 longin domain.

Original languageEnglish
Pages (from-to)397-417
Number of pages21
JournalBiology of the Cell
Volume104
Issue number7
DOIs
StatePublished - Jul 2012

Keywords

  • Cell cycle
  • Intracellular compartmentalisation
  • Protein sorting/trafficking/targeting
  • Secretion
  • Vesicle trafficking

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