title: R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed.
abstract: BACKGROUND
  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 ∼100 nm 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.
authors.affiliation: Division of Biological Sciences and Centre for Structural and Functional Neuroscience, The University of Montana, Missoula, MT 59812, USA.
authors.forename: Nandhakumar
authors.initials: N
authors.lastname: Thayanidhi
authors.forename: Yingjian
authors.initials: Y
authors.lastname: Liang
authors.forename: Haruki
authors.initials: H
authors.lastname: Hasegawa
authors.forename: Deborah C
authors.initials: DC
authors.lastname: Nycz
authors.forename: Viola
authors.initials: V
authors.lastname: Oorschot
authors.forename: Judith
authors.initials: J
authors.lastname: Klumperman
authors.forename: Jesse C
authors.initials: JC
authors.lastname: Hay
mesh_terms: D000818:Animals; D002453:Cell Cycle; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D048429:Cell Size; D004583:Electrons; D008940:Mitotic Index; D008954:Models, Biological; D015388:Organelles / Q000378:metabolism; D016716:PC12 Cells; D010447:Peptide Hydrolases / Q000378:metabolism*; D020836:Protein Structure, Quaternary; D050683:R-SNARE Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013194:Staining and Labeling
chemical_list: D050683:R-SNARE Proteins; C491481:Ykt6 protein, rat; D010447:Peptide Hydrolases