The life cycle of the human immunodeficiency virus (HIV) and other retroviruses is dependent upon nuclear transport. Once in the cytoplasm, HIV RNA is reverse-transcribed into double-stranded DNA that must enter the nucleus. The viral regulatory proteins, Rev and Tat, have been identified as key modulators of the transcription and transport of HIV envelope mRNA out of the nucleus. We are examining the processes of nuclear protein import and export both in vivo and in vitro using the HIV Rev protein. The Rev protein actively shuttles between nucleolus and cytoplasm and mediates premature export of unspliced retroviral RNAs. Using digitonin permeabilized cultured cells, we developed an in vitro assay for studying both nuclear import and export as well as nucleolar accumulation of molecules such as HIV Rev. These data suggest that dissociation from the nucleolus is an ATP-dependent process. Utilizing Fluorescence Recovery After Photobleaching, Rev was found to be immobilized at the nucleolus, further strengthening the role for an ATP-dependent release from the nucleolus. This regulated nucleolar localization may reflect a more widespread phenomenon since it has been observed for other nucleolar proteins involved in apoptosis and cell cycle control. The system we have described should allow for the identification of the requirements for regulated nucleolar targeting, as well as nuclear transport of other shuttling proteins. Currently we are characterizing the physiological role of calreticulin in glucocorticoid recepor export. The nuclear pore complex mediates all traffic between the nucleus and cytoplasm. This complex contains proteins that are highly decorated at serine and theronine residues by N-acetyl glucosamine. The exact role of this type of glycosylation is unclear. As it shares many of the characteristics of phosphorylation, it is thought to represent a type of signalling mechanism. Our laboratory has cloned and expressed the enzymes responsible for the addition (O-linked GlcNAc Transferase, OGT) and removal (O-GlcNAcase) of this sugar moiety. These enzymes are currently being used in conjunction with our in vivo and in vitro assay for nuclear transport (see above) to determine the exact role of the glycosylated nuclear pore complex in nuclear import and export.