Our laboratory studies macromolecular transport into and out of the nucleus in eukaryotic cells. The high degree of compartmentalization in eukaryotes causes a spatial separation of cellular processes such as DNA transcription and mRNA translation. This leads to the bi-directional exchange of a large number of macromolecules between the nuclear and cytoplasmic compartments and allows additional levels of control in eukaryotic gene expression. The site of exchange is the nuclear pore complex (NPC), a multiprotein structure, consisting of probably more than 100 different polypeptides. Every minute several thousand macromolecules are transported through a single NPC in both directions. Export cargoes include different classes of RNA such as messenger RNA (mRNA) , ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNAs (snRNAs). In the opposite direction, a large number of proteins and matured snRNP particles are actively imported into the nucleoplasm. In addition, a number of proteins have been shown to shuttle between the nucleus and the cytoplasm. Despite recent progress in our understanding of nucleocytoplasmic transport processes many fundamental questions remain. For example, the export of mRNA from the nucleus into the cytoplasm, a key step in the control of eukaryotic gene expression, is still very poorly understood. Also, the actual mechanism how macromolecules are translocated through the NPC is largely unknown. We combine genetic and biochemical approaches in S. cerevisiae and metazoan cells to characterize the molecular machinery responsible for the transport of macromolecules through the NPC.
Export of macromolecules from the nucleus. Export of RNA and proteins from the nucleus into the cytoplasm is a fundamental cellular process and a key step in the control of eukaryotic gene expression. Kinetic competition experiments indicate that different RNA classes, including mRNA, snRNA, tRNA and rRNA use distinct export pathways. Since most, if not all RNAs, are associated with proteins in the nucleus it is thought that RNA export events are mediated by proteins containing appropriate nuclear export signals (NESs). Our laboratory analyzes and characterizes RNA and protein export pathways in the yeast Saccharomyces cerevisisiae. We have identified the nuclear export factor exportin 1 (Xpo1p/Crm1p) as a carrier of nuclear export signal (NES)-mediated protein export in S. cerevisiae. In addition to the role in protein transport, mutations in XPO1 have also been shown to affect the export of poly-adenylated RNA. Using a variety of approaches we characterize the molecular function of exportin 1. We also continue to dissect the pathway that targets leucine-rich NES-containing proteins from the nucleus to the cytoplasm. We have chosen this export pathway as a model system in our aim to further understand how macromolecules are translocated through the NPC into the cytoplasm.
Mechanism of protein import into the nucleus. Four soluble factors have been previously identified and characterized that mediate the uptake of nuclear localization signal (NLS)-containing proteins into the nucleus in an in vitro import system. These factors comprise the heterodimeric NLS-receptor (importin a/b), the GTPase Ran and the small homodimeric protein NTF2/p10. Importin b interacts directly with proteins of the NPC and this interaction is regulated by the RanGTP/GDP cycle. It is not well understood, how GTP hydrolysis by Ran is mechanistically coupled to the translocation of proteins and multiprotein complexes through the roughly 150nm-long aqueous channel of the NPC. With the help of in vitro import systems we try to further dissect and understand the molecular mechanism of this cellular transport pathway.