We established a straightforward experimental system to investigate directly the requirements for nucleocytoplasmic transport in live cells. For this purpose, substrates were created containing nuclear localization signals (NLS) or nuclear export signals (NES) linked to a chimeric protein composed of the glutathione S-transferase (GST) fused to the green fluorescent protein (GFP). The combinati...

Labeled nuclear proteins were microinjected into the cytoplasm of Tetrahymena thermophila. Macronuclear H1, calf thymus H1, and the SV40 large T antigen nuclear localization signal linked to BSA accumulated specifically in macronuclei, even if cells were in micronuclear S phase or were nonreplicating. The way in which histone H4 localized to either the macronucleus or the micronucleus suggested...

SUMOylation, reversible attachment of small ubiquitin-related modifier (SUMO), serves to regulate hundreds of proteins. Consistent with predominantly nuclear targets, enzymes required for attachment and removal of SUMO are highly enriched in this compartment. This is true also for the first enzyme of the SUMOylation cascade, the SUMO E1 enzyme heterodimer, Aos1/Uba2 (SAE1/SAE2). This essential ...

The nuclear import of the spliceosomal snRNPs U1, U2, U4 and U5, is dependent on the presence of a complex nuclear localization signal (NLS). The latter is composed of the 5'-2,2,7-terminal trimethylguanosine (m3G) cap structure of the U snRNA and the Sm core domain. Here, we describe the isolation and cDNA cloning of a 45 kDa protein, termed snurportin1, which interacts specifically with m3G-c...

A convenient assay for monitoring nuclear localization signal-mediated nuclear import of green fluorescent protein (GFP)-variant fusion proteins has been developed. This modified assay relies upon indirect immunofluorescence microscopy for visualization of transported substrates. The use of GFP-variant fusion proteins allows for the rapid assessment of optimal digitonin concentration and permit...

La (SS-B) is a highly expressed protein that is able to bind 3'-oligouridylate and other common RNA sequence/structural motifs. By virtue of these interactions, La is present in a myriad of nuclear and cytoplasmic ribonucleoprotein complexes in vivo where it may function as an RNA-folding protein or RNA chaperone. We have recently characterized the nuclear import pathway of the S. cerevisiae La...

Importin alpha plays a pivotal role in the classical nuclear protein import pathway. Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway. In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfami...

Rel-family transcription factors function in a variety of biological processes, including development and immunity. During early Drosophila development, the Toll-Cactus-Dorsal pathway regulates the establishment of the embryonic dorsoventral axis. The last step in this pathway is the graded nuclear import of the Rel protein Dorsal. Dorsal is retained in the cytoplasm by the IkappaB-family prote...

Kap104p is a Saccharomyces cerevisiae nuclear import receptor for two essential mRNA-binding proteins, Nab2p and Nab4p/Hrp1p. We demonstrate direct binding of Kap104p to each of these substrates. We have defined the nuclear localization signals in both Nab2p and Nab4p/Hrp1p by Kap104p binding in vitro and KAP104-dependent nuclear import in vivo. The nuclear localization signals map to similar a...