Transcript MB207_12 - MB207Jan2010

MB 207 – Molecular Cell Biology
Intracellular
compartments and
protein sorting
Compartmentalization of cells
• Bacterium consists of a single intracellular compartment surrounded by plasma
membrane. Eukaryotic cell is subdidvided into functionally distinct, membraneenclosed compartments.
• Each compartment or organelle contains its own characteristic of enzymes and
other specialized molecules, and complex distribution systems transport specific
products from one compartment to another.
→ protein (enzymes, transporters, surface markers)
• 10 000 – 20 000 proteins are synthesized in the cytosol and delivered
specifically to the cell compartment that requires it.
Major intracellular compartments of an animal cell
→ Vital biochemical processes take place in or on membrane surfaces.
→ Compartments increasing surface area as well as providing
specialized aqueous spaces for reaction.
• In most cells, Golgi apparatus
is located close to the nucleus
whereas the network of ER
tubules extends from the
nucleus throughout the entire
cytosol.
• The localization of both ER
and Golgi apparatus depends
on an intact microtubule array.
The evolution of internal membranes: Development of plastid
Hypothetical scheme for the evolution of the cell nucleus and ER
Topological relationships between compartments of the secretory
and endocytic pathways in eucaryotic cell
• Cycles of membrane budding and fusion permit the lumen of any of these organelles to
communicate with any other and with cell exterior by means of transport vesicles.
• Arrows indicate the extensive network of outbound and inbound traffic routes.
A simplified ‘roadmap’ of protein traffic
• Gated transport: Gated system
(nuclear pore complexes) to actively
transport specific molecules.
• Transmembrane transport: Membranebound protein translocaters directly
transport proteins across a membrane.
• Vesicular transport: Membraneenclosed vesicles transport proteins
from one compartment to another.
Three main mechanisms of protein transport
A
A
B
B
C
C
Vesicular transport: Vesicular budding and fussion
Summarizes the routes by which protein are
carried forward or diverted to other organelles
General principles about protein trafficking
•
Signal sequences and signal patches direct proteins to the correct cellular
location
- Signal sequence: signal resides in a single discrete stretch of amino acid
sequence, often cleaved, not part of final protein product
- Signal patch: 3-D arrangements of amino acids on the protein’s surface that
forms when the protein folds up
•
Proteins synthesized on free cytoplasmic ribosomes are imported posttranslationally (e.g. nuclei, mitochondria, peroxisomes) and proteins
synthesized on ER-bound ribosomes are imported co-translationally (e.g. ER)
•
Proteins can fold before membrane translocation if translocation machinery can
cope with large, folded protein structures (e.g. nuclei & peroxisomes) OR they
will fold only after membrane translocation (e.g. mitochondria & ER).
Signal sequence and signal patch
The transport of molecules between nucleus and
cytosol
→ Bidiectional traffic occurs continuously between cytosol and nucleus.
Nuclear pore complexes perforate the nuclear envelope
• composed of more than 50 nucleoporins, arranged in octagonal
symmetry.
• more active the nucleus in transcription, greater the number of pore
complexes.
• On average, each pore need to import 100 histone molecules/min and
export 6 large and small ribosomal subunits/min.
Possible paths for free diffusion through nuclear pore complex
• Results from injection (molecules of different sizes)
→ <5000 daltons molecule: fast diffusion
17kD protein: 2 mins
>60kD: cannot enter
→ channel is 9nm in diameter and 15nm long
• Nuclear envelope enables nuclear compartment and cytosol to maintain
different complements of proteins.
→ eg. protein synthesis is confined to the cytosol.
Nuclear localization signals direct nuclear proteins to the nucleus
Immunofluorescence micrographs showing T-antigen localization.
Nuclear import receptors bind nuclear localization signals and
nucleoporins
A. Many nuclear import receptors bind both to nucleoporins and to a nuclear
localization signal on the cargo proteins they transport. Cargo proteins 1, 2
and 3 contain different nuclear localization signals, which causes each to
bind to a different nuclear import receptor.
B. Cargo protein 4 requires as adaptor protein to bind its nuclear import
receptor. The adaptors are structurally related to nuclear import receptors
and recognize nuclear localization signals on cargo proteins. They also
contain a nuclear localization signal that binds them to an import receptor.
• Nuclear export receptors relies on nuclear export signals to transport
macromolecules to cytosol.
The Ran GTPase drives directional transport
• Ran is required for both nuclear import and export systems.
• Ran is a molecular switch that can exist in two conformational states,
depending on whether GDP or GTP is bound.
Ran-GTP causes cargo
binding of export
receptor.
Ran-GTP causes cargo release in
the nucleus and GTP-bound
receptors return to cytosol.
Nuclear localization of activated T cells control gene
expression
Nuclear lamina
• Is a meshwork of interconnected protein subunits called nuclear
lamins.
→ Lamins are special class of intermediate filament proteins that
polymerize into a two-dimensional lattice.
• Gives shape and stability to the nuclear envelope, interact with
nuclear pore complexes, integral proteins and chromatin.
The breakdown and reformation of the nuclear envelope
during mitosis
NLS is not cleaved off
after transport into
nucleus.
The transport of proteins into mitochondria and
chloroplasts
• In contrast to the cristae of mitochondria, the
thylakoids of chloroplasts are not connected to the
inner membrane and therefore form a
compartment with a separate internal space.
• Mainly depending on the import of proteins from
the cytosol.
→ proteins need to be translocated.
Translocation into mitochondrial matrix depends on a signal
sequence and protein translocators
Signal Sequence
• at N terminus, rapidly removed after
import by a protease (signal peptidase) in
matrix.
• folded into an amphipathic α helix,
positively charged (red) clustered on one
side of the helix while uncharged
hydrophobic (yellow) are clustered
primarily on the opposite face.
Protein translocators
• TOM complex: translocase for outer membrane
TIM complex: translocase for inner membrane
• Both complexes contain components that act as receptors for mitochondrial precursor
proteins and other components that form the translocation channel.
• OXA complex:
- protein translocator in the inner mitochondrial membrane.
- mediates the insertion of inner membrane proteins that synthesized within the
mitochondria.
- helps to insert some protein that are initially transported into the matrix by TOM and TIM
complexes.
Protein import by mitochondria
• TOM complex first transports the mitochondrial targeting signal across the
outer membrane.
• Reaches in the intermembrane space, targeting signals binds to a TIM
complex, opening the channel in the complex through which the polypeptide
chain either enters the matrix.
• The signal sequence is cleaved off by a signal peptidase in the matrix to form
the mature protein. The free signal sequence is then rapidly degraded.
ATP hydrolysis and a H+ gradient are used to drive protein import
into mitochondria
(1) Bound cytosolic hsp70 is released from the protein in a step that depends on
ATP hydrolysis. After the initial insertion of the signal sequence and of adjacent
portions of the polypeptide chain into TOM complex, the signal sequence interacts
with a TIM complex. (2) The signal sequence is then translocated into the matrix in a
process that requires
Two plausible models of how mitochondrial hsp70 could drive
protein import
• In the thermal ratchet
model, the translocating
polypeptide chain slides
back and forth, driven by
thermal motion, and it is
successively trapped in the
matrix by hsp70 binding.
• In the cross-bridge
ratchet model, a
conformational change in
hsp70 actively pulls the
chain into the matrix.
Protein import from the cytosol into the inner mitochondrial
membrane or intermembrane space
Translocation of a precursor protein into thylakoid space of
chloroplasts
• Two signal sequences are
required for proteins
directed to thylakoid
membrane in chloroplasts.
• Signal sequences for
mitochondria and
chloroplasts are diffferent.
• Four routes of
translocation into thylakoid
membrane
Peroxisomes
• Contain high concentrations of oxidative
enzymes (catalase and urate oxidase).
• Major sites of oxygen utilization.
• Use molecular oxygen to remove
hydrogen atoms from specific organic
substrates (R) in an oxidative reaction
that produces hydrogen peroxide.
RH2 + O2 → R + H2O2
• Catalase utilizes H2O2
• Surrounded by a single membrane and
do not contain DNA or ribosomes.
• Acquire their proteins by selective import
from the cytosol.
A model for how new peroxisomes are produced
• A short signal sequence (3
amino acids located at the Cterminus or near N-terminus)
directs the import proteins into
peroxisomes.
• Peroxisomes are thought to form only form preexisting peroxisomes by
a process of growth and fission.
Endoplasmic reticulum
• ER membrane separates the ER lumen from cytosol
and mediates the selective transfer of molecules
between these two compartments.
• play central role in lipid and protein biosynthesis.
• captures proteins from the cytosol
i) transmembrane proteins which are partly
translocated across the ER membrane and become
embeddedin it.
ii) water-soluble proteins which are fully translocated
across the ER membrane and are released into the ER
lumen.
• Ribosome is directly attached to the ER membrane.
→ synthesizing protein.
• Two spatially separate populations of ribosomes in
the cytosol:
i) membrane-bound ribosomes
ii) free ribosomes
Free and membrane-bound ribosomes
• A common pool of ribosomes is
used to synthesize the proteins that
stay in the cytosol and those that are
transported into the ER.
• The ER signal sequence on a
newly formed polypeptide chain
directs the engaged ribosome to the
ER membrane.
• The mRNA molecule remains
permanently bound to the ER as
part of a polyribosome, while the
ribosomes that move along it are
recycled.
• At the end of each round of protein
synthesis, the ribosomal subunits
are released and rejoin the common
pool in the cytosol.
The signal hypothesis: protein translocation across the ER membrane
• When the ER signal sequence emerges from the ribosome, it directs the ribosome to
a translocator on the ER membrane that forms a pore in the membrane through which
the polypeptide is translocated.
• The signal sequence is clipped off during translation by a signal peptidase, and the
mature protein is released into the lumen of the ER immediately after being
synthesized.
The signal-recognition particle (SRP)
(A) Mammalian SRP is an elongated complex containing six subunits and one
RNA molecule (SRP RNA). One end of the SRP binds to an ER signal
sequence on a growing polypeptide chain, while the other binds to the
ribosome itself and pauses translation. The RNA in the particle may mediate
an interaction with ribosomal RNA.
(B) The crystal structure of the signal-sequence-binding domain of a bacterial
SRP subunit. The domain contains a large, exposed binding pocket that is
lined by hydrophobic amino acids, a large number of which are methionines.
The outline of the pocket is shaded in gray to emphasize its location. The
flexible side chains of methionine are ideal for building adaptable
hydrophobic binding sites for other proteins.
How ER signal sequences and SRP direct ribosomes to the ER
membrane
• The SRP binds to both the exposed ER signal sequence and the ribosome, thereby inducing a
pause in translation.
• The SRP receptor in the ER membrane, which it is composed of two different polypeptide
chains, binds the SRP-ribosome complex and directs it to the translocator.
• The SRP and SRP receptor are then released, leaving the ribosome bound to the translocator
in the ER membrane.
• The translocator then inserts the polypeptide chain into the membrane and transfers it across
the lipid bilayer.
• SRP release occurs only after the ribosome has become properly engaged with the
translocator in the ER membrane.
• The translocator is closed until the ribosome has bound, so that the permeability barrier of the
ER membrane is maintained at all times.
Three ways in which protein translocation can be driven through
structurally similar translocators
A model for how a soluble protein is translocated across ER
membrane
• Upon binding an ER signal sequence, the translocator opens its pore, allowing the
transfer of the polypeptide chain across the lipid bilayer as a loop.
• After the protein has been completely translocated, the pore closes, but the translocator
now opens laterally within the lipid bilayer, allowing the hydrophobic signal sequence to
diffuse into the bilayer, where it is rapidly degraded.
How a single-pass transmembrane protein with a cleaved ER
signal sequence is integrated into the ER membrane
• Co-translational translocation process is initiated by an N-terminal ER sequence that
functions as a start-transfer signal.
• In addition to this start-transfer, the protein also contains a stop-transfer sequence.
• When the stop-transfer sequence enters the translocator and interacts with a binding site, the
translocator changes its conformation and discharges the protien laterally into the lipid bilayer.
Protein glycosylation in the rough ER
• Almost as soon as a polypeptide
chain enters the ER lumen, it is
glycosylated on target asparagine
amino acids.
• The precursor oligosaccharide is
transferred to the asparagine as
an intact unit in a reaction
catalyzed by a membrane-bound
oligosaccharyl transferase
enzyme.
• One copy of this enzyme is
associated with each protein
translocator in the ER membrane.
The role of N-linked glycosylation in ER protein folding
• The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins
containing one terminal glucose on N-linked oligosaccharides, trapping the protein in the ER.
• Removal of the terminal glucose by a glucosidase releases the protein from calnexin.
• A glucosyl transferase is the crucial enzyme that determines whether the protein is folded
properly or not. If the protein is still incompletely folded, the enzyme transfers a new glucose
from UDP-glucose to the N-linked oligosaccharide, renewing the protein’s affinity for calnexin
and retaining it in the ER.
• The cycle repeats until the protein has folded completely.
The export and degradation of misfolded ER proteins
• Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they
are deglycosylated, ubiquitylated and degraded in proteasomes.
• Misfolded membrane proteins follow a similar pathway. Misfolded proteins are exported
through the same type of translocator that mediated their import; accessory proteins that are
associated with the tranlocator allow it to operate in the export direction.
The unfolded protein response in yeast
Phospholipid exchange proteins help to tranport phospholipids
from the ER to mitochondria and peroxisomes
• Phospholipids are insoluble in water, their passage between membranes requires carrier
proteins.
• Phospholipid exchange proteins are water-soluble proteins that carry a single molecule of
phospholipid at a time. They can pick up a lipid molecule from one membrane and release it at
another, thereby redistributing phospholipids between membrane-enclosed compartments.
• The net transfer of phosphatodycholine (PC) from the ER to mitochondria can occur without
the input of additional energy because the concentration of PC is high in the ER membrane
and low in the outer mitochondrial membrane.