Mammalian SR proteins are currently thought to function in mRNA export as w
ell as splicing. They contain multiple phosphorylated serine/arginine (RS/S
R) dipeptides. Although SR domains can be phosphorylated by many kinases in
vitro, the physiologically relevant kinase(s), and the role(s) of these mo
difications in vivo have remained unclear. NpI3 is a shuttling protein in b
udding yeast that we showed previously to be a substrate for the mammalian
SR protein kinase, SRPK1, as well as the related yeast kinase, Sky1, Here w
e demonstrate that Sky1p phosphorylates only one of NpI3p's eight SR/RS dip
eptides. Mutation of the C-terminal RS to RA, or deletion of SKY1, results
in the cytoplasmic accumulation of NpI3p, The redistribution of Npl3p is ac
companied by its increased association with poly(A)(+) RNA and decreased as
sociation with its import receptor, Mtr10p, in vivo. We propose that phosph
orylation of NpI3p by the cytoplasmically localized Sky1p is required for e
fficient release of mRNA upon termination of export.