Pseudophosphorylated αB-Crystallin Is a Nuclear Chaperone Imported into the Nucleus with Help of the SMN Complex

The human small heat shock protein alpha B-crystallin (HspB5) is a molecular chaperone which is mainly localized in the cytoplasm. A small fraction can also be found in nuclear speckles, of which the localization is mediated by successional phosphorylation at Ser-59 and Ser-45. alpha B-crystallin does not contain a canonical nuclear localization signal sequence and the mechanism by which alpha B-crystallin is imported into the nucleus is not known. Here we show that after heat shock pseudophosphorylated alpha B-crystallin mutant alpha B-STD, in which all three phosphorylatable serine residues (Ser-19, Ser-45 and Ser-59) were replaced by negatively charged aspartate residues, is released from the nuclear speckles. This allows alpha B-crystallin to chaperone proteins in the nucleoplasm, as shown by the ability of alpha B-STD to restore nuclear firefly luciferase activity after a heat shock. With the help of a yeast two-hybrid screen we found that alpha B-crystallin can interact with the C-terminal part of Gemin3 and confirmed this interaction by co-immunoprecipitation. Gemin3 is a component of the SMN complex, which is involved in the assembly and nuclear import of U-snRNPs. Knockdown of Gemin3 in an in situ nuclear import assay strongly reduced the accumulation of alpha B-STD in nuclear speckles. Furthermore, depletion of SMN inhibited nuclear import of fluorescently labeled recombinant alpha B-STD in an in vitro nuclear import assay, which could be restored by the addition of purified SMN complex. These results show that the SMN-complex facilitates the accumulation of hyperphosphorylated alpha B-crystallin in nuclear speckles, thereby creating a chaperone depot enabling a rapid chaperone function in the nucleus in response to stress.