Transcript d05a1663be3edc4

Alberts • Johnson • Lewis • Raff • Roberts • Walter
Molecular Biology of the Cell
Fifth Edition
Chapter 12
Intracellular Compartments and
Protein Sorting
Copyright © Garland Science 2008
Membrane Enclosed Organelles in a Eukaryotic cell
Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)
Table 12-1 Molecular Biology of the Cell (© Garland Science 2008)
Table 12-2 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-2 Molecular Biology of the Cell (© Garland Science 2008)
Evolution of membranes  specialization of function
Figure 12-3a Molecular Biology of the Cell (© Garland Science 2008)
Development of thylakoid in chloroplasts
Figure 12-3b Molecular Biology of the Cell (© Garland Science 2008)
Origin of compartments in Eukaryotic cells
Figure 12-4a Molecular Biology of the Cell (© Garland Science 2008)
Origin of compartments in Eukaryotic cells
Figure 12-4b Molecular Biology of the Cell (© Garland Science 2008)
Topological relationship between compartments
Figure 12-5 Molecular Biology of the Cell (© Garland Science 2008)
Proteins can move between compartments in different ways
Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)
Proteins can move between compartments in different ways
• synthesis of all proteins begins on ribosomes in the cytosol of
Eukaryotic cells (except on mitochondria)
• their fate depends on their amino acid sequence, which can contain
a sorting sequence that directs their delivery to locations outside the
cytosol.
• If no sorting sequence is present the protein stays in the cytosol.
How proteins move from one compartment to another
• Gated transport : between nucleus and cytoplasm; via nuclear pore
complex (selective gate for macromolecules).
• transmembrane transport : from cytosol into a space that is
topologically distinct. Via protein translocators (proteins unfold in
translocators).
• vesicular transport : transport vesicles ferry proteins from one
compartment to the next. Between topologically equivalent areas.
How proteins move from one compartment to another:
VESICULAR TRANSPORT
Figure 12-7 Molecular Biology of the Cell (© Garland Science 2008)
Sorting Sequences Direct Proteins to the Correct Cell Address
• signal sequences (ss):
• 15-60 amino acids long
• often found at the N-terminus of the protein
• signal peptidase enzyme removes the ss after the protein arrives
to its destination.
• can also be internal stretches of amino acids, these remain part
of the protein.
• sometimes they are made of a 3D arrangement of amino acids
called a signal patch.
• Each ss specifies a particular destination in the cell
• are necessary and sufficient for protein targeting
• recognized by sorting receptors that guide them to appropriate
location. Receptors are reused (like a bus).
Sorting Sequences Direct Proteins to the Correct Cell Address
Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)
Panel 12-1 important
Most organelles can not be formed de novo, they need information from
the organelle itself
• During cell division, most organelles become tiny vesicles, so
that when the cell divides in two it has in it some of each
organelle that reforms. So the information to make the organelle
is not in the DNA but it is in the organelle itself.
Page 704 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear envelop organization
Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear pore complexes in the nuclear envelop
Figure 12-9 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear pore complexes proteins are nucleoporins
Figure 12-9a Molecular Biology of the Cell (© Garland Science 2008)
Nuclear pore complexes (NPCs)
• In a cell there are 3000-4000 NPCs
• Transports up to 500 macromolecules per second
• Can transport in both directions at the same time
• Each contain one or more aqueous passages : small water soluble
molecules can pass by passive diffusion. (5000 daltons or less) (each
aa is ~110 daltons
Figure 12-9b Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-9c Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-9d Molecular Biology of the Cell (© Garland Science 2008)
Nuclear pore complexes: size cut-off to free diffusion
Figure 12-10 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear Localization Signals (NLS) directs nuclear proteins to the
nucleus
Figure 12-11 Molecular Biology of the Cell (© Garland Science 2008)
Visualizing active import through the NPC: coating gold particles with
NLS signal.  Electron microscopy
Figure 12-12 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear import receptor bind to both NPCs and to NLS
Figure 12-13 Molecular Biology of the Cell (© Garland Science 2008)
Nuclear import receptors
• The import receptors are soluble cytosolic proteins
• The import receptors bind to the NPC at FG repeats
• The receptor-cargo complexes move along the transport path
by repeatedly binding, dissociating and re-binding to adjacent
FG-repeats.
• Once inside the nucleus the receptor dissociates from cargo
and return to the cytosol.
Nuclear export works like nuclear import, but in reverse
• Nuclear Export Signals and Nuclear Export Receptors are
needed for export.
• The Nuclear Export Receptors are called Karyopherins.
The Ran GTPase Imposes Directionality on Transport Through the
NPCs
Figure 12-14 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-15 Molecular Biology of the Cell (© Garland Science 2008)
Binding of Ran-GTP causes the import receptor to release their cargo
Figure 12-16a Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-16b Molecular Biology of the Cell (© Garland Science 2008)
There is a tight regulation in the proteins localization in/out of the
nucleus
Figure 12-17 Molecular Biology of the Cell (© Garland Science 2008)
Cells conrol transport by regulating NL an NE signals; these can be
turned on/off by phosphorylation of residues near the signal sequence
Figure 12-18 Molecular Biology of the Cell (© Garland Science 2008)
The nuclear lamina is located on the nuclear side of the inner nuclear
membrane: a meshwork of interconnected proteins called nuclear lamins:
lamins are intermediate filaments.
Figure 12-19 Molecular Biology of the Cell (© Garland Science 2008)
During mitosis the nuclear envelope breaks down
Figure 12-20 Molecular Biology of the Cell (© Garland Science 2008)
• When nucleus disassembles during mitosis, the nuclear lamina
depolymerizes.
• The disassembly is a result of direct phosphorylation of the nuclear
lamins by a kinase called cyclin dependent kinase (cdk) that is
activated at mitosis.
• later in mitosis the nuclear envelope reassembles on the surface of
chromosomes.
• NLS are not cleaved off after import presumably because they need
to be imported repeatedly once after cell division.
Page 723 Molecular Biology of the Cell (© Garland Science 2008)
The endoplasmic reticulum
• membrane constitutes more than half of the total membrane in
animal cells.
• It consists of branching tubules and flattened sacs
• Its membrane is continuous with the nuclear envelope
• ER lumen is called cisternal space
• It serves as calcium storage
• All proteins that are sent outside the cell, or to the golgi or to
lysosomes pass by the ER first.
Figure 12-34a Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-34b Molecular Biology of the Cell (© Garland Science 2008)
ER functional specialization
• Rough ER
In mammalian cells proteins are imported into the ER before
complete synthesis of the polypeptide chain – co-translational
translocation (whereas in mitochondria and peroxisomes it is posttranslational).
• Rough ER has ribosomes on it
• Smooth ER does not have ribosomes; involved in lipid
metabolism, streroid.
• Transitional ER are areas of SER where vesicles bud off
Co- and post- translational translocation into the ER
Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-36a Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-36b Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)
ER functional specialization – liver cells
• In the liver most cells are hepatocytes, have ;large amount of smooth
ER. This makes lipoprotein particles which carries lipid in the blood to
other parts of the body.
• Also present on ER membranes are enzymes that detoxify lipid
soluble drugs, the enzyme called cytochrome P450 makes drugs that
are water insoluble, water soluble, so now they are excreted in the
urine.
• Also the ER functions in Calcium storage; in response to extracellular
signals calcium is released in the cytoplasm (ex. Muscle cells
sacroplasmic reticulum)
ER derived vesicles = microsomes are used to study the ER
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)
Purified Rough ER microsomes by electron microscopy
Figure 12-37a Molecular Biology of the Cell (© Garland Science 2008)
Protein translocation into the ER
• Transmembrane proteins partially translocate across the ER
membrane.
• Water soluble proteins fully translocate and are released in the
lumen of the ER
 The proteins are directed by the ER Signal Sequence
Signal hypothesis
• The experiment that showed the function of ER signal sequence:
mRNA translated in vitro to make a normally secreted protein; without
microsomes this protein is larger than normal (secreted); it was found
that it has an N-terminal leader peptide, which gets cleaved off by
signal peptidase in the ER after translocation…
Signal hypothesis
Figure 12-38 Molecular Biology of the Cell (© Garland Science 2008)
ER Signal Sequence and SRP
• Guided by SRP: Signal Recognition Particle which cycles between the ER
membrane and the cytosol;
• SRP binds to the ER SS and to a receptor on the ER membrane.
• SRP consists of 6 proteins + 1 RNA molecule
• ER SS vary greatly in aa sequence; but has 8 or more non-polar aa in its center
• So how can the SRP be so specific to all the different SS?
Has a hydrophobic pocket.
• SRP is rod-like; one end it binds ER SS another end bonds between the large and
small ribosomal subunits  blocks elongation, stoping translation; this ensures that
the protein is not sent to the cytoplasm
(this is VERY important for lysosomal enzymes as they can be deadly if the cell
puts them in the cytoplasm)
SRP
Figure 12-39a Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-39b Molecular Biology of the Cell (© Garland Science 2008)
SRP binds to ER SS and to SRP receptor followed by targeting to
translocator in the ER membrane
Figure 12-40 Molecular Biology of the Cell (© Garland Science 2008)
Two pools of ribosomes (cytoplasmic and ER associated)
Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-41b Molecular Biology of the Cell (© Garland Science 2008)
The translocator (sec61 complex) forms a water filled pore in the
membrane: polypeptide chain passes through this pore
Figure 12-42 Molecular Biology of the Cell (© Garland Science 2008)
In Eukaryotes four sec61 complexes assemble to form a large
translocator that can be visualized on ribosomes
Figure 12-43 Molecular Biology of the Cell (© Garland Science 2008)
• in post-transational translocation BiP, a chaperone assists proteins in
folding in the lumen of the ER, actually providing the pulling force to
help enter proteins in the ER. This drives un-directional transport
• no energy is needed in co-translational translocation
Figure 12-44 Molecular Biology of the Cell (© Garland Science 2008)
• ER SS triggers opening of the translocator: it is
recognized twice, once by RSP and second time by the
translocator – here it acts as “start-transfer signal”
•When the nascent (new) polypeptide chain grows long
enough ER signal peptidase cleaves off the signal
sequence (that gets degraded by proteases in the ER
membrane)
Figure 12-45 Molecular Biology of the Cell (© Garland Science 2008)
Single pass transmembrane protein
• N-ter SS initiates translocation, but an additional
hydrophobic segment in polypeptide chain stops
the transfer process before the entire chain is
translocated: stop-transfer signal anchors the
protein n the membrane (12-46)
• other cases when SS is internal here is stays as
the transmembrane domain (12-47)
Single pass transmembrane protein
Figure 12-46 Molecular Biology of the Cell (© Garland Science 2008)
Single pass transmembrane protein
Figure 12-47 Molecular Biology of the Cell (© Garland Science 2008)
Multipass transmembrane proteins
• Polypeptide passes back and fourth repeatedly
across the lipid bilayer; here there is a start transfer
sequence followed by a stop transfer sequence
• In more complex proteins they have many start
transfer sequences and many stop transfer
sequences
Multipass transmembrane proteins
Figure 12-48 Molecular Biology of the Cell (© Garland Science 2008)
Multipass transmembrane protein
Figure 12-49 Molecular Biology of the Cell (© Garland Science 2008)
ER resident proteins
• Contain ER retention signal (4 amino acids in C-terminus)
• One important ER resident protein is protein disulfide isomerase
(PDI): catalyzes oxidaion of free –SH groups to form disulfide –SS- bonds
• Another ER resident protein is BiP chaperone: pulls proteins posttranslationally into the ER through the translocator.
Most proteins synthesizes in the RER are
glycosylated by the addition of N-linked
oligosacharides
• covalent addition of sugars
• most soluble proteins in ER are glycoproteins
• very few proteins in the cytosol are glycosylated
• N-linked (N is asparagine); these sugars are added to asparagine
Set of sugars is added en bloc by oligosaccharyl
transferase : 2 N-acetylglucoseamine, 9 mannose, 3
glucose
Figure 12-50 Molecular Biology of the Cell (© Garland Science 2008)
Dolichol holds the precursor oligosaccharide in ER
membrane
Figure 12-51 Molecular Biology of the Cell (© Garland Science 2008)
Synthesis of the lipid linked precursor oligosaccharide in the
ER membrane
Nucleotide-sugar
intermediates
High energy Phosphate
bonds
Figure 12-52 Molecular Biology of the Cell (© Garland Science 2008)
• N-linked oligosaccharides are most common in ER
• following this, there is glucose trimming (2 glucose taken
out)
• Less frequently oligosaccharides are linked to the
hydroxyl group on the side chain of a serine, threonine or
hydroxylysine : theese are O-linked in the Golgi
• Some proteins require N-linked oligosaccharides for
proper folding; calnexin and calreticulin are lectins, they
are chaperones, they bind to the sugar groups of
improperly folded proteins and retain them in the ER. 
unfolded proteins have glucose added to them and
removed continuously (by glucosidase and glycosyl
transferase)
Incompletely folded proteins are retained in the ER until they
fold properly
Figure 12-53 Molecular Biology of the Cell (© Garland Science 2008)
When a protein does not fold properly and exits the ER, it is
degraded by the proteasome; sugar is removed and ubiquitin is
added
Figure 12-54 Molecular Biology of the Cell (© Garland Science 2008)
Cells carefully monitor eth amount of misfolded proteins
• in the ER misfolded proteins trigger the “unfolded protein response”:
• Increase transcription of genes encoding ER chaperones, proteins
involved in transport from ER to cytosol (retrotranslocation) and
protein degradation in the cytosol.
HOW IS THIS DONE?
How a misfolded protein send a signal to make more
chaperones
Figure 12-55a Molecular Biology of the Cell (© Garland Science 2008)
How a misfolded protein send a signal to make more
chaperones
Figure 12-55b Molecular Biology of the Cell (© Garland Science 2008)
Some membrane proteins acquire a covalently attached
Glycosylphosphatidylinositol (GPI) anchor
• GPI is added to the C-ter of proteins destined to the plasma membrane,
also into lipid rafts. These can then be released from the GPI anchor by
specific phospholipases in the plasma membrane
Figure 12-56 Molecular Biology of the Cell (© Garland Science 2008)
The ER assembles most lipid bilayers: phosphatidylcholine,
one major phospholipid
Figure 12-57 Molecular Biology of the Cell (© Garland Science 2008)
Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)