Transcript Conclusion: a) The nuclear localization signal (NLS)

From Cytosol to organelles---post-translational
translocation
The big picture :
From Cytosol to the nucleus
The road map: nucleus structure
1) The envelope: this is the barrier that separates N from cytosolic
space. Inner and outer membrane: the outer membrane is
connected with ER, perinuclear space connected with ER lumen.
2) Nuclear pores
Very large protein
complex of more
than 100 different
proteins forms a
basket-like
structure through
the double
membranes
The pore complex in plant cells is less well studied but it has been
show to contain glycoproteins (probed by lectins that bind sugar
groups)
3) Diffusion and “active” transport through the pore:
Small molecules are freely diffusing back and forth but molecules
larger than 60 KD need active transport and require signal sequence
for getting in.
4) Two way traffic! Proteins get in and RNAs get out
2. The signal sequence and the mechanism of import
1) Nuclear localization signal (NLS)
Classical experiments using frog oocytes and microinjection: gold particles
coated with a nuclear protein—dissecting the signal sequence/domain
Head
tail
tail
Head
Many procedures can be used to study the signal sequences that are
responsible for nuclear import: in vitro experiments using isolated
cells or nucleus and in vivo experiments using transgenic plants or
transiently transformed cells—monitoring the presence of your
protein of interest (by labeling them with fluorescence or other
detectable signal).
Conclusion: a) The nuclear localization signal (NLS) often consists of two
patches of positively charged amino acids (4 or more residues in each
patch). This pattern is not very strictly enforced. b) Such sequence can
direct other proteins or even gold particles into the nucleus---no photo ID
required. C) Not cleaved!
XXXX(R/K)nXXXXXXXX (R/K)n
2) Receptors for NLS:
In vitro assay of nuclear transport: permeabilized animal culture
cells---plus the labeled protein---does not go into the nucleus
even when NLS is present---plus ATP and cell lysate (cytoplasmic
components)---goes to nucleus:
Conclusion: ATP and cytoplasmic proteins/factors are required
for import. Using this assay, the soluble factors were purified as
importin alpha and beta and a small G protein called Ran. The
importins form dimeric complex that recognize NLS and bind the
target protein. IN the presence of ATP, importin beta interacts
with nuclear pore complex (NPC) and they get into the nucleus.
What is Ran doing? Ran is shown to facilitate the dissociation of
cargo from importin complex and the export of importin beta for
the shuttling process. The proteins GAP and GEF are GTPase
activation protein (GAP) and guanine nucleotide exchange factor
(GEF). They regulate the activity of Ran.
3) Regulation of the import traffic:
Control of protein import provides another mechanism for cell
regulation—protein is produced but may not be imported
into the nucleus—who has the priority to enter?
This control can be performed by many possible ways such as
protein phosphorylation or dephosphorylation, protein
complex formation, and so on. These processes can be
tightly controled by presence of signals including hormones
or environmental cues. In animal cells, a classical example
is steroid receptor import. In plant cells, light-regulated
protein import—COP1 repressor protein is present in both
cytosol and nucleus in light but only in nucleus in the dark.
Gene activation
steroids
NLS
NLS
hsp90
NLS
hsp90
3. Nuclear Export
1) What to be exported? mRNA as an example.
mRNA in the nucleus is packaged by proteins forming
ribonucleoproteins (RNPs).
2) Nuclear export signal (NES): is in the proteins that
associate with the mRNA. The details of such
signals are not clear because several different
sequences have been shown to serve as NES in
different proteins—more diverse than the NLS.
3) The receptors: exportins
The receptors for the NES sequences are called
exportins (vs importins for import). The cycle of
exportin action is quite similar to the importin
cycle and also involves Ran, the small G protein.
Comparing the import and
export:
a) some components like
Ran are shared.
b) They also share the
same pores (one pore
can handle two way
traffic)
c) But “tickets” and
receptors are different
Other export activities: the mechanisms for other types of
RNAs are less understood