Vaults are dynamically unconstrained cytoplasmic nanoparticles capable of half vault exchange.
Vaults are naturally occurring ribonucleoprotein particles with an enormous interior volume, large enough to encapsulate hundreds of proteins. They are highly conserved and are present in nearly all eukaryotic cells ranging from 10(4) to 10(7) particles per cell. Recombinant vaults can be produced in vitro and engineered to allow cell targeting and protein packaging. These nanometer-sized particles have many desirable characteristics that may give them advantages for use as drug delivery vehicles. Using photoactivatable green fluorescent protein (PAGFP) labeled vaults, we demonstrate that the particles rapidly diffuse throughout the cytoplasm following single pixel photoactivation in live cells. Their in vivo movement remained relatively unchanged despite exposure to a variety of cellular stresses, suggesting that vaults are largely unconstrained in the cytoplasm. Fluorescence resonance energy transfer (FRET) was observed from polyethylene glycol (PEG) fused hybrid cells that expressed either CFP or YFP labeled vaults, indicating that vaults can exchange major vault protein (MVP) subunits in vivo. Investigation into the mechanism of this exchange in vitro using recombinant vaults demonstrated that they were capable of rapidly separating at the particle waist and reassembling back into whole vaults, supporting a half vault exchange mechanism. This data suggests a means whereby vaults can functionally interact with their cellular environment and deliver materials packaged within their interior.
Department of Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, California 90095, USA.
Jian
J
Yang
Valerie A
VA
Kickhoefer
Benny C
BC
Ng
Ajaykumar
A
Gopal
Laurent A
LA
Bentolila
Scott
S
John
Sarah H
SH
Tolbert
Leonard H
LH
Rome
D000818:Animals; D045744:Cell Line, Tumor; D003593:Cytoplasm / Q000378:metabolism*; D004058:Diffusion; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D053758:Nanoparticles; D011092:Polyethylene Glycols / Q000737:chemistry; D011487:Protein Conformation; D021381:Protein Transport; D053838:Scattering, Small Angle; D020394:Vault Ribonucleoprotein Particles / Q000737:chemistry* / Q000378:metabolism*; D014961:X-Ray Diffraction; D000099187:Major Vault Protein
D011092:Polyethylene Glycols; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein