FRAP analysis of nucleocytoplasmic dynamics of the vitamin D receptor splice variant VDRB1: preferential targeting to nuclear speckles.
Although the key components of the cellular nuclear transport machinery have largely been characterized through extensive efforts in recent years, in vivo measurements of the kinetics of nuclear protein import/export are patently few. The present study applies the approach of FRAP (fluorescence recovery after photobleaching) to examine the nucleocytoplasmic flux of a novel human VDRB1 (vitamin D receptor B1) isoform in living cells. Through an N-terminal extension containing a consensus nuclear targeting sequence, VDRB1 is capable of localizing in nuclear speckles adjacent to SC-35 (35 kDa splicing component)-containing speckles as well as in the nucleoplasm, dependent on ligand. Investigation of VDRB1 nucleocytoplasmic transport using FRAP indicates for the first time that the VDRB1 has a serum-modulated, active nuclear import mechanism. There is no evidence of an efficient, active export mechanism for VDRB1, probably as a result of nuclear retention. VDRB1 nuclear import in the absence of serum occurred more rapidly and to a greater extent to nuclear speckles compared with import to other nuclear sites. This preferential transport from the cytoplasm to and accumulation within nuclear speckles is consistent with the idea that the latter represent dynamic centres of VDRB1 interaction with other nuclear proteins. The results are consistent with the existence of specialized pathways to target proteins to nuclear subdomains.
Sunn
Kathryn L
KL
Bone and Mineral Program, Garvan Institute of Medical Research, Sydney, NSW 2010, Australia.
Eisman
John A
JA
Gardiner
Edith M
EM
Jans
David A
DA
D021581:Active Transport, Cell Nucleus; D000818:Animals; D019556:COS Cells; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D036681:Fluorescence Recovery After Photobleaching; D042581:Intranuclear Space / Q000378:metabolism; D018613:Microscopy, Confocal; D020033:Protein Isoforms / Q000378:metabolism; D017434:Protein Structure, Tertiary; D018167:Receptors, Calcitriol / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D044967:Serum
D020033:Protein Isoforms; D018167:Receptors, Calcitriol; D011993:Recombinant Fusion Proteins