All retroviruses must circumvent cellular restrictions on the export of unspliced RNAs from the nucleus. While the unspliced RNA export pathways for HIV and Mason-Pfizer monkey virus are well characterized, that of Rous sarcoma virus (RSV) is not. We have previously reported that the RSV direct repeat (DR) elements are involved in the cytoplasmic accumulation of unspliced viral RNA. Here, using fluorescent in situ hybridization (FISH), we demonstrate that unspliced viral RNAs bearing a single point mutation (G8863C) in the DR exhibit a restricted cellular localization in and around the nucleus. In contrast, wild type unspliced viral RNA had a diffuse localization throughout the nucleus and cytoplasm. Since the RSV Gag protein has a transient localization in the nucleus, we examined the effect of Gag over-expression on a DR-mediated reporter construct. While Gag did not enhance DR-mediated nuclear export, the dominant-negative expression of two cellular export factors, Tap and Dbp5, inhibited expression of the same reporter construct. Furthermore, FISH studies using the dominant-negative Dbp5 demonstrated that unspliced wild type RSV RNA was retained within the nucleus. Taken together, these results further implicate the DR in nuclear RNA export through interactions with Tap and Dbp5.

The luminal domains of membrane peptidylglycine ␣-amidating monooxygenase (PAM) are essential for peptide ␣-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion. INTRODUCTIONAtT-20 corticotrope tumor cells have long served as a reliable model system for studying the biosynthesis, storage, and regulated secretion of pituitary peptide hormones (Mains and Eipper, 1978;Moore and Kelly, 1986;Tooze and Tooze, 1986). Cleavage of endogenous pro-opiomelanocortin (POMC) by PC1 (PC1) and carboxypeptidase E yields both adrenocorticotropic hormone (ACTH) and ␤-lipotropin (Fricker and Devi, 1993;Zhou et al., 1993). Production of other POMC peptides requires the action of peptidylglycine ␣-amidating monooxygenase (PAM) (Eipper et al., 1986). Although levels of PAM in AtT-20 cells are 20-fold lower than in the anterior pituitary, complete amidation of POMCderived products occurs (Eipper et al., 1986).PAM is one of the few peptide-processing enzymes that spans the secretory granule membrane. The fact that its luminal, catalytic domains are pH sensitive and are further activated on cleavage from the membrane (Husten and Eipper, 1991;Husten et al., 1993) raised the possibility that PAM might play a role in signaling luminal conditions to the cytosolic machinery involved in secretory granule formation. This type of signaling is essential in communicating information about events occurring in the lumen of the endoplasmic reticulum to cytosolic proteins (Pahl and Baeuerle, 1997;Shamu, 1997;Brown and Goldstein, 1998), as well as in cargo selection during vesicle budding (Kuehn and Herrmann, 1998).We were surprised to find that expression of exogenous PAM in AtT-20 cells at levels equivalent to those in the anterior pituitary blocked the regulated secretion of ACTH, Abbreviations used: ACTH, adrenocorticotropic hormone; DC, COOH-terminal domain of PAM; CHO, C...