The HECT ubiquitin ligase Rsp5p is required for proper nuclear export of mRNA in Saccharomyces cerevisiae.

The nuclear transport of both proteins and RNAs has attracted considerable interest in recent years. However, regulation pathways of the nuclear transport machineries are still not well characterized. Previous studies indicated that ubiquitination is involved in poly(A)+ RNA nuclear export. For this reason, we systematically investigated ubiquitin-protein ligasess from the homologous to E6-AP carboxy terminus (HECT) family for potential individual roles in nuclear transport in Saccharomyces cerevisiae. Here we report that Rsp5, an essential yeast ubiquitin ligase involved in many cellular functions, when deleted or mutated in ligase activity, blocks the nuclear export of mRNAs. Affected messenger RNAs include both total poly(A)+ mRNA and heat-shock mRNAs. Mutation of Rsp5 does not affect nuclear protein import or export. Deletion of RSP5 blocks mRNA export, even under conditions where its essential role in unsaturated fatty acids biosynthesis is bypassed. Using domain mapping, we find that the ligase activity is required for proper mRNA export, indicating that ubiquitination by Rsp5 acts directly or indirectly to affect RNA export. The finding that Rsp5p ligase mutations cause a more pronounced defect at high temperatures suggests that ubiquitination of transport factors by Rsp5p may also be essential during stress conditions.

Protein export from the nucleus.

The evolution of the nucleus imposed on eukaryotic cells the necessity to strictly control exchange of molecules between the nucleus and the remainder of the cell, not only to protect and correctly transmit genetic information, but also to coordinate nuclear and cytoplasmic functions. Studies over the past 10 years have provided major insights into the molecular mechanisms responsible for transport of molecules between the nucleus and the cytoplasm. In addition, regulation of the nucleocytoplasmic distribution of diverse cellular factors has emerged as one of the most efficient mechanism to adapt gene expression to the cell environment, for example by controlling the access of transcriptional regulators to their target genes. In this review, we focus on the molecular basis of protein nuclear export that relies on interactions between targeting sequences present on the cargoes, specific export receptors or exportins and nuclear pore proteins, with special emphasis on the role of the Ran GTPase and associated proteins in this process.

Terminal minihelix, a novel RNA motif that directs polymerase III transcripts to the cell cytoplasm. Terminal minihelix and RNA export.

Determining the cis-acting elements controlling nuclear export of RNA is critical, because they specify which RNA will be selected for transport. We have characterized the nuclear export motif of the adenoviral VA1 RNA, a small cytoplasmic RNA transcribed by RNA polymerase III. Using a large panel of VA1 mutants in both transfected COS cells and injected Xenopus oocytes, we showed that the terminal stem of VA1 is necessary and sufficient for its export. Surprisingly, we found that the nucleotide sequence within the terminal stem is not important. Rather, the salient features of this motif are its length and its relative position within the RNA. Such stems thus define a novel and degenerate cytoplasmic localization motif that we termed the minihelix. This motif is found in a variety of polymerase III transcripts, and cross-competition analysis in Xenopus oocytes revealed that export of one such RNA, like hY1 RNA, is specifically competed by VA1 or artificial minihelix. Taken together these results show that the minihelix defines a new cis-acting export element and that this motif could be exported via a novel and specific nuclear export pathway.

NXT1 is necessary for the terminal step of Crm1-mediated nuclear export.

Soluble factors are required to mediate nuclear export of protein and RNA through the nuclear pore complex (NPC). These soluble factors include receptors that bind directly to the transport substrate and regulators that determine the assembly state of receptor-substrate complexes. We recently reported the identification of NXT1, an NTF2-related export factor that stimulates nuclear protein export in permeabilized cells and undergoes nucleocytoplasmic shuttling in vivo (Black, B.E., L. Lévesque, J.M. Holaska, T.C. Wood, and B.M. Paschal. 1999. Mol. Cell. Biol. 19:8616-8624). Here, we describe the molecular characterization of NXT1 in the context of the Crm1-dependent export pathway. We find that NXT1 binds directly to Crm1, and that the interaction is sensitive to the presence of Ran-GTP. Moreover, mutations in NXT1 that reduce binding to Crm1 inhibit the activity of NXT1 in nuclear export assays. We show that recombinant Crm1 and Ran are sufficient to reconstitute nuclear translocation of a Rev reporter protein from the nucleolus to an antibody accessible site on the cytoplasmic side of the NPC. Further progress on the export pathway, including the terminal step of Crm1 and Rev reporter protein release, requires NXT1. We propose that NXT1 engages with the export complex in the nucleoplasm, and that it facilitates delivery of the export complex to a site on the cytoplasmic side of NPC where the receptor and substrate are released into the cytoplasm.

Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease.

The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion. To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus. Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm. In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin. The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed. The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+. These results suggest that loop 4-5 is near or part of the cation binding site. Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone. The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation.

In vivo membrane assembly of the E.coli polytopic protein, melibiose permease, occurs via a Sec-independent process which requires the protonmotive force.

To investigate the mechanism of polytopic membrane protein insertion in Escherichia coli, we have examined the protein and energy requirements for in vivo membrane assembly of the prototypic 12 transmembrane domain sugar co-transporter, melibiose permease (MelB). MelB membrane assembly was analyzed both kinetically, by pulse labeling experiments, and functionally by measuring the activity of the inserted permease. Strikingly, the rate of MelB membrane assembly is decreased approximately 4-fold upon dissipation of the transmembrane electrochemical proton gradient, delta(mu)H+, indicative of a strong requirement for delta(mu)H+. Interestingly, selective dissipation of either the electrical (delta(psi)) or the chemical (delta(pH)) component of delta(mu)H+ demonstrates that either form of energy is required for MelB membrane assembly. In contrast, MelB membrane assembly does not require SecA, SecY or SecE, all three proteins which are strictly required for protein translocation. Neither the rate of MelB membrane assembly nor the amount of functional permease is affected by inactivation or depletion of these Sec proteins. These results strongly suggest that polytopic membrane proteins such as MelB insert into the cytoplasmic membrane by a mechanism fundamentally different from protein translocation.