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.
Current Projects
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.
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