Vault mobility depends in part on microtubules and vaults can be recruited to the nuclear envelope.
Vaults are ribonucleoproteins that may function in intracellular transport processes. We investigated the intracellular distribution and dynamics of vaults in non-small cell lung cancer cells in which vaults are labeled with the green fluorescent protein. Immunofluorescence experiments showed that vaults are dispersed throughout the cytoplasm; a small fraction is found in close proximity to microtubules. Immunoprecipitation experiments corroborated these results showing co-precipitation of MVP and beta-tubulin. Using quantitative fluorescence-recovery after photobleaching (FRAP), we demonstrated that vault mobility over longer distances in part depends on intact microtubules; vaults moving slower when microtubules are depolymerized by nocodazole. Biochemical fractionation indicated a small fraction of MVP associated with the nucleus, however, no GFP-tagged vaults could be observed inside the nucleus. We observed an accumulation of vaults at the nuclear envelope upon treatment of cells with the protein synthesis inhibitor cycloheximide. Analysis of nucleo-cytoplasmic transport using a fluorescent substrate containing a classical NLS and NES expressed in MVP+/+ and MVP-/- mouse embryonic fibroblasts indicated no differences in nuclear import/export kinetics, suggesting no role for vaults in these processes. We hypothesize that a subset of vaults moves directionally via microtubules, possibly towards the nucleus.
van Zon
Arend
A
Department of Hematology, Erasmus Medical Center, PO Box 1738, 3000 DR Rotterdam, The Netherlands.
Mossink
Marieke H
MH
Houtsmuller
Adriaan B
AB
Schoester
Martijn
M
Scheffer
George L
GL
Scheper
Rik J
RJ
Sonneveld
Pieter
P
Wiemer
Erik A C
EA
D021581:Active Transport, Cell Nucleus; D001692:Biological Transport; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000378:metabolism; D005347:Fibroblasts / Q000502:physiology; D036681:Fluorescence Recovery After Photobleaching; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009685:Nuclear Envelope / Q000378:metabolism*; D011485:Protein Binding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011510:Proteolipids / Q000235:genetics / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000502:physiology; D014404:Tubulin / Q000378:metabolism; D014407:Tumor Cells, Cultured; D025262:Vacuolar Proton-Translocating ATPases; D020394:Vault Ribonucleoprotein Particles / Q000235:genetics / Q000378:metabolism*; D000099187:Major Vault Protein
D003513:Cycloheximide; D049452:Green Fluorescent Proteins; D011500:Protein Synthesis Inhibitors; D011510:Proteolipids; D006180:Proton-Translocating ATPases; D014404:Tubulin; D025262:Vacuolar Proton-Translocating ATPases; D020394:Vault Ribonucleoprotein Particles; D000099187:Major Vault Protein; C437713:Atp6v0c protein, mouse; C485184:enhanced green fluorescent protein