12. Transport of proteins synthesized on free ribosomes. Nuclear envelope, transport through nuclear pores

Question 1

Protein importin mitochodria, nuclear envelope

Answer 1

protein import into mitochondria
Mitochondrial proteins synthetized in the cytoplasm have an N-terminal mitochondrial
targeting (signal) sequence which directs them into the matrix. They are imported in unfolded
form across protein channels spanning both mitochondrial membranes (TIM and TOM proteins,
translocase of the inner /outer mitochondrial membrane). During the process cytoplasmic and
matrix hsp70 heat shock proteins (chaperons) keep the proteins in unfolded conformation, and
hsp60 molecule complexes facilitate the formation of their final tertiary structure in the matrix.
The structure of mitochondrial hsp60 is similar to that of the bacterial heat shock proteins.
nuclear envelope
The nuclear envelope is composed of two concentric lipid bilayers termed the inner and outer
nuclear membranes and in higher eukaryotes also of the nuclear lamina connected to the inner
membrane. The outer membrane is continuous with the endoplasmic reticulum, and ribosomes
can be found on its outer surface. The inner and outer nuclear membranes are separated by a
perinuclear space that is continuous with the lumen of the endoplasmic reticulum. Nuclear pores
span the two membranes connecting the cytosol with the nucleoplasm.

Question 2

nuclear lamina, nuclear pore

Answer 2

nuclear lamina
A protein meshwork composed of intermediate filaments called lamins beneath the inner
nuclear membrane that mechanically supports the nuclear membrane. It plays an important role
in maintaining the structure and stability of nuclear pores, and it is involved in the organisation
of chromatin inside the nucleus. (Lamin B binds to the inner membrane, while lamin A and C
bind both to lamin B and the chromatin.)
nuclear pores, NPC (nuclear pore complex)
A large molecular complex composed of nucleoporins (approximately 100 proteins in
mammals), that spans the double nuclear membrane. It ensures bidirectional, selective transport
between the cytoplasm and the nucleus. The central channel of the nuclear pore has a diameter
of about 100 nm and it is surrounded by 8 smaller peripheral channels. Cytoplasmic filaments
and protein fibres composing the nuclear basket play a role in the docking of transported
molecules upon their nucleo-cytoplasmic transport. The nuclear envelope of a typical
mammalian cell contains 5-7000 nuclear pores.

Question 3

nuclear porine, nuclear importina

Answer 3

nucleoporins
Proteins forming the nuclear pore complex (approximately 50-100 proteins in mammals) are
called nucleoporins. Certain nucleoporins contain unstructured side-chains composed of repeats
of phenylalanine-glycine dipeptides (FG repeats). These side-chains protrude into the lumen of
the pores forming a meshwork that acts as a filter: small molecules are let to pass through, but
larger molecules (>50-60 kDa) can penetrate only if they have a nuclear localization signal
(NLS) that is recognized by importins.
importins
Importins are transport receptor protein which facilitates the import of nuclear proteins (cargo
molecules) into the nucleus through the nuclear pore complex. Importin beta either binds to the
NLS of the cargo directly, or if that is not possible, through the importin α adapter protein.
Interaction of importin beta with the cytoplasmic fibrils of the pore complex (=docking) and
with the phenylalanine-glycine (FG)-rich side-chains of nucleoprins facilitates the import of
cargo molecules through the pores.

Question 4

RAn GDP, GTP, NLSnuclear localization signal

Answer 4

Ran-GTP and Ran-GDP gradient
Ran, a small (monomeric) G protein, is involved in the synchronization of nuclear transport
process. Localization of Ran-GDP to the cytosol and Ran-GTP to the nucleus results from the
localization of two Ran regulatory proteins: Ran GTPase-activating protein (Ran-GAP) is
located in the cytosol and Ran guanine nucleotide exchange factor (Ran-GEF) is bound to
chromatin and is hence exclusively found in the nucleus. Ran-GTP or Ran-GDP binding to the
transporter determines the direction of transport and initiates it across the nuclear pore.
NLS (nuclear localization signal)
The selectivity of the nuclear import process is based on the nuclear localization signals (NLSs),
which are present only in nuclear proteins. NLS is a (7-10 amino acid long) sorting signal
(motif) and it is recognized by importin molecules on the cargo molecules. The signal can be
located anywhere in the linear sequence of the protein, but it has to be recognisable for importins
on the surface of the folded protein. NLS of some proteins is exposed upon reversible
posttranslational modification, such as phosphorylationm. The recogniton of NLS is necessary
and sufficient for nuclear import.

Question 5

NES
Ran gap
Ran gep cc2

Answer 5

NES (nuclear export signal)
Proteins exported from the nucleus bear a nuclear export signal (e.g. a leucine-rich sequence)
that is recognized by transport receptors called exportins.
Ran-GAP
A GTPase activating protein localized in the cytoplasm (it is anchored to the cytoplasmic fibrils
of the pore complex). Ran-GAPs stimulate the GTPase activity of Ran, and GTP hydrolysis
occurs immediately when Ran arrives to the cytosol, therefore the overwhelming majority of
Ran is in GDP-bound form in the cytoplasm.
Ran-GEF (RCC1)
Ran must release its bound GDP to acquire GTP. GDP dissociation from Ran is slow but is
stimulated by a nucleotide exchange factor (guanine exchange factor) named Ran-GEF, which
is exclusively localized in the nucleus, and is tightly associated with chromatin throughout the
cell cycle. Therefore, Ran molecules are always in GTP-bound form in the nucleus. Exchange
of GDP for GTP promotes the dissociation of importin from its trasported cargo protein.