In eukaryotes, the multivesicular body (MVB) sorting pathway plays an essential role in regulating cell surface protein composition, thereby impacting numerous cellular functions. Vps4, an ATPase associated with a variety of cellular activities, is required late in the MVB sorting reaction to dissociate the endosomal sorting complex required for transport (ESCRT), a requisite for proper function of this pathway. However, regulation of Vps4 function is not understood. We characterize Vta1 as a positive regulator of Vps4 both in vivo and in vitro. Vta1 promotes proper assembly of Vps4 and stimulates its ATPase activity through the conserved Vta1/SBP1/LIP5 region present in Vta1 homologues across evolution, including human SBP1 and Arabidopsis thaliana LIP5. These results suggest an evolutionarily conserved mechanism through which the disassembly of the ESCRT proteins, and thereby MVB sorting, is regulated by the Vta1/SBP1/LIP5 proteins.

The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.

Despite the apparent overall structural stability of the nuclear pore complex during interphase, at least two nucleoporins have been shown to move dynamically on and off the pore. It is not yet certain what contribution nucleoporin mobility makes to the process of nuclear transport or how such mobility is regulated. Previously, we showed that Nup98 dynamically interacts with the NPC as well as bodies within the nucleus in a transcription-dependent manner. We have extended our studies of dynamics to include Nup153, another mobile nucleoporin implicated in RNA export. In both cases, we found that although only one domain is essential for NPC localization, other regions of the protein significantly affect the stability of association with the pore. Interestingly, like Nup98, the exchange of Nup153 on and off the pore is inhibited when transcription by Pol I and Pol II is blocked. We have mapped the regions required to link Nup98 and Nup153 mobility to transcription and found that the requirements differ depending on which polymerases are inhibited. Our data support a model whereby transcription of RNA is coupled to nucleoporin mobility, perhaps ultimately linking transport of RNAs to a cycle of remodeling at the nuclear pore basket.

Separation of the nucleus and cytoplasm, maintained by two membrane bilayers that form the nuclear envelope, allows for spatial control over transcription factors and signaling molecules. This compartmentalization further ensures the presence of specialized environments for different stages of gene expression, such as transcription and protein production. Selective exchange between these two compartments is clearly important as well. Whereas many types of active transport between the nucleus and cytoplasm rely on transport receptors in the importin-␤ superfamily, export of mRNA utilizes distinct soluble machinery (6, 92). Moreover, in general mRNA export does not depend on a specific motif in the cargo, as has been demonstrated in many other cases of receptor-cargo interactions (22). Recent progress in identifying soluble factors important to mRNA trafficking is beginning to reveal the molecular basis for functional coupling between steps in mRNA biogenesis and how such coupling, rather than a consensus motif, brings specificity to mRNA export.Studies with Saccharomyces cerevisiae revealed that the key modulators of cellular mRNA export are unrelated to canonical importin-␤-related receptors. Specifically, yeast deficient in a gene called MEX67 accumulate poly (A) ϩ RNA in the nucleus (73). A second protein, Mtr2p, binds Mex67p, and this interaction is required for the export of poly(A) ϩ RNA in yeast (71,77). In an independent avenue of investigation, involving metazoan cells and the simian type D retrovirus Mason Pfizer monkey virus, the cellular protein TAP was found to facilitate export of RNA containing the viral constitutive transport element (CTE) (8, 32). TAP, confirmed to be the human orthologue of Mex67p, has been redesignated NXF1 (nuclear export factor 1).NXF1 interacts with p15/NXT1, the presumed functional homologue of Mtr2p (33,44). Although Mtr2p and p15 share no sequence similarity, the Mex67p-Mtr2p complex displays similar structural architecture to the NXF1-p15 heterodimer (21). Indeed, the mRNA export defect in yeast cells deficient in both Mex67p and Mtr2p can be rescued by expression of human NXF1 and its cofactor p15 (44). Expression knock-down studies using RNA interference have demonstrated that NXF1 and p15 are required for poly(A) ϩ RNA export (35,84,93), further strengthening the case for an evolutionarily conserved system of mRNA export that is distinct from the importin-␤ superfamily. Likewise, studies with Xenopus oocytes support the conclusion that the small GTPase Ran, a key modulator of importin-␤-type receptors, is not key to mRNA export (10). However, the distinction between mRNA export and the importin-␤ family/Ran network is not absolute, as an importin-␤ family member has recently been implicated in mRNA export as well (74). mRNA EXPORT IS COUPLED TO SPLICINGIn contrast to CTE-mediated transport, NXF1 does not bind directly to cellular mRNA. Therefore, deciphering how NXF1 is recruited to mRNA is crucial to understanding mRNA export and has been the focus of much current ...