BIOL 266 – CELL BIOLOGY
Lecture 9:
Protein sorting to organelles (II) –
secretory pathway
nucleus Proteins are sorted from the ER onward
and from one compartment of the
endomembrane system to another
Proteins are transported by transport
vesicles
Transport vesicles become loaded with a
chloroplast
cargo of proteins from the interior space
(lumen) of one compartment, as they pinch
mitochondrion off from its membrane
These transport vesicles subsequently
peroxisome
ER discharge their cargo into a second
lysosome compartment by fusing with its membrane
Golgi
apparatus
3 TRANSPORT BY VESICLES
Proteins remain folded during the transport
Pinches off Fuses
Folded protein Transport vesicle
A major outward pathway of vesicular transport (traffic) –
the secretory pathway
IMPORT
lysosome
Golgi apparatus
ER plasma membrane
external
medium
Secretory pathway: Proteins are transported from the ER, through the Golgi
apparatus, to the lysosome, plasma membrane or external medium
Two branches of the secretory pathway:
(1) lysosomal branch
(2) exocytic branch (aka exocytosis)
(1) lysosomal branch
IMPORT lysosome
Golgi apparatus
ER (2) exocytosis
Each of the membrane-bound compartments of the
endomembrane system communicates with one another
by means of transport vesicles
Different types of transport vesicles:
IMPORT lysosome
Golgi apparatus
ER
To perform its function correctly, each transport vesicle that buds
off from a compartment must:
(1) take with it only the proteins appropriate to its destination
(2) fuse only with the appropriate target membrane
IMPORT lysosome
Golgi apparatus
ER
1st example: A vesicle carrying cargo proteins
from the ER to the Golgi apparatus must:
(1) exclude protein(s) that are to stay in the ER
(2) take with it only proteins appropriate to the Golgi
(3) fuse only with with the Golgi apparatus membrane
IMPORT lysosome
2nd example: A vesicle carrying cargo protein(s) from the
trans Golgi network to the lysosome must:
(1) exclude protein(s) that are to stay in the Golgi
(2) exclude proteins that are to be transported to the plasma
membrane and external medium
(3) take with it only protein(s) appropriate to the lysosome
(4) fuse only with with the lysosomal membrane
IMPORT
lysosome
trans Golgi network
Due to this selectivity of the processes of vesicle packaging, budding and
fusion:
Each organelle of the endomembrane system maintains its own distinct
identity, that is, its own distinctive protein composition
ER: contains only protein Golgi: contains only protein
Lysosome: contains only Plasma membrane: contains only
IMPORT
lysosome
trans Golgi network
Newly synthesized proteins destined to reside in organelles of the
endomembrane system are co-translationally imported to the rough
ER as unfolded, monomeric and unmodified polypeptides
Their folding, assembly into multimeric complexes and covalent
modification occur in the rough ER
Co-translational
import
SRP
Translocon
SRP receptor
ER
Loop of the ribosome-bound polypeptide chain
Newly synthesized proteins in the membrane and lumen of the
ER undergo five principal modifications before they reach their
final destinations, various organelles of the endomembrane
system:
(1) formation of disulfide bonds SH HS S S
(2) addition and processing of carbohydrates
(3) specific proteolytic cleavage
(4) proper folding
(5) assembly into multimeric proteins + +
Disulfide bonds are formed in the lumen of the rough ER but not in the cytosol
Cystein Cystein
Sulfhydryl Interchain
CH2 groups CH2 disulfide
bond
SH S
oxidants + enzyme
SH reductants S
CH2 CH2
CH2 CH2
SH S
Intrachain
disulfide
SH S bond
CH2 CH2
CYTOSOL: reducing environment, ER: oxidative environment,
high level of glutathione GSH low level of glutathione GSH
Disulfide bonds are found only in secretory proteins and in
the extracellular domains of plasma membrane proteins
Function of disulfide bonds formation in the ER:
Help to stabilize the structure of those proteins that may
encounter changes in pH and degradative enzymes after
they are incorporated into the plasma membrane
Extracellular
CELL medium
pH pH can be
homeostasis changed (no pH
SH HS S S homeostasis)
SENSITIVE to RESISTANT to
changes in pH changes in pH Proteases are not
Proteases are
and to proteases and to proteases surrounded by
inside
membranes
lysosomes
secretory
extracellular domains proteins
of PM proteins
Most plasma membrane and secretory proteins that initially enter the
ER lumen or ER membrane contain one or more carbohydrate chains
The covalent attachment of short carbohydrate chains converts these
proteins into glycoproteins
This process of glycosylation is carried out by glycosylating enzymes
found in the ER but not in the cytosol
Glycosylases Glycoprotein
(oligosaccharide Glucose
protein transferases)
Galactose
Mannose
CYTOSOL: ER: several
glycosylases are oligosaccharide-
not found and protein-
specific
glycosylases are
present
N-linked protein glycosylation in the ER
a specialized
N-acetylglucosamine mannose glucose dolichol membrane lipid
Translocon
Cytosol
dolichol
dolichol
e
gin
ER lumen
a
P
ar
Asparagine
p
Loop of the
As
P ribosome-bound P
NH2 growing
N
polypeptide chain
Oligosaccharide protein transferase
As soon as a polypeptide chain enters the ER lumen, it is
glycosylated by covalent attachment of a preformed
branched oligosaccharide to particular asparagines in
the polypeptide
Functions of oligosaccharides covalently attached to proteins in the ER
(1) protect the protein from degradation:
Sensitive to proteases in Resistant to proteases in
the extracellular medium the extracellular medium
(2) retain the protein in the ER until it properly folded:
Unfolded or misfolded protein:
unable to exit the ER, binds to
oligosaccharide-specific chaperone + chaperone
Properly folded protein:
able to exit the ER
(3) help guide the protein to the appropriate organelle by serving as a
transport signal for packaging the protein into appropriate transport vesicles
ER ER vesicle
Correct folding of newly made proteins is facilitated by several ER
chaperones, proteins that accelerate the folding within the ER lumen
STEP 5
The ER chaperone protein Hsp70 uses
release of the ribosome
the energy of ATP hydrolysis:
(1) to pull the protein inside, into the
ER lumen
(2) to assist in the final folding of the
translocated protein into its mature,
active state
Pulling Hsp70 ATP
inside ADP Hsp70 ATP
ADP
Folding
Folding and assembly of proteins into multimeric protein complexes
is assisted by two ER chaperones, Calnexin and Calreticulin
Calreticulin
1st step: 2nd step: 3rd step:
Protein glycosylation (1) binding to Calnexin (1) release of the
and Calreticulin ribosome
Calnexin
(2) formation of three (2) assembly of a
Protein: Hemagglutinin disulfide bonds trimeric protein
1st mechanism that controls protein exit from the ER: ER-resident
proteins are retained in the ER by an ER retention signal
budding
ER transport
vesicle
ER retention signal: a Proteins that are able to exit from the ER:
carboxyl-terminal
tetrapeptide KDEL = (1) do not contain the ER retention signal
Lys-Asp-Glu-Leu (2) properly folded
KDEL receptor: (3) properly assembled into multisubunit
protein complexes
(1) binds to the ER retention signal
(2) acts to retain ER-resident proteins in the ER
(3) acts to retrieve proteins with the KDEL sequence
that have escaped to the cis-Golgi network and return
them to the ER
2nd mechanism that controls protein exit from the ER:
Quality control in the ER
ER
2 1 budding
transport
vesicle
chaperone
export
Properly folded monomeric proteins and
degradation properly assembled non-resident
multimeric protein complexes are able to
exit from the ER
Misfolded or unassembled non-resident proteins: Are actively
retained in the ER lumen by binding to chaperones
2 1
Proteins that fail to fold or assemble properly: Interaction with chaperones holds
(1) are exported from the ER to the cytosol misfolded and unassembled proteins in the
(2) are then degraded in the cytosol ER until proper folding and assembly occur
3rd mechanism that controls protein exit from the ER: the assembly
of a protein coat on the cytosolic surface of transport vesicles
budding budding
vesicle vesicle
ER ER
cytosolic coat proteins
protein budding
Vesicles that bud from the ER and other coat
membranes of organelles of the endomembrane
system have a distinct protein coat on their coated
cytosolic surface and therefore called coated vesicle
vesicles
After budding is completed, the coat is lost, uncoating
allowing the membrane of the vesicle (vesicular
membrane) to interact directly with the
membrane to which it will fuse (target budding
vesicle
membrane)
Two functions of protein coat on the cytosolic surface of budding vesicles:
(1) it shapes the donor membrane into a bud
(2) it helps to capture only cargo proteins (membrane-bound and soluble)
into budding vesicles
membrane cargo protein
ER-resident protein
KDEL receptor
budding
vesicle
ER (donor
membrane)
soluble cargo protein
Components that participate in budding of coated vesicles
small GTP-
1st step:
binding protein
Budding is initiated by
soluble cargo recruitment of a small
protein GTP-binding protein
from the cytosol to the
cytosolic surface of a
donor membrane
membrane
cargo protein
This small GTP-
binding protein
donor
membrane regulates the rate of
vesicle formation
Components that participate in budding of coated vesicles
Adapter for the
membrane cargo protein
Adapter for the soluble
cargo protein
2nd step:
Coat and adapter
proteins are recruited
from the cytosol to the
cytosolic surface of the
budding vesicles
coat protein
Components that participate in budding of coated vesicles
Signal sequence that is recognized by an
adapter for the membrane cargo protein
3rd step:
Adapter proteins select which
membrane and soluble proteins
will enter the transport vesicles
as cargo proteins
These cargo proteins contain
short signal sequences that are
recognized by the corresponding
Signal sequence that is recognized by an
adapter for the soluble cargo protein adapters and direct them to a
specific type of transport vesicle
Components that participate in budding of coated vesicles
polymerized coat subunit proteins
4th step:
The coat subunit proteins
polymerize around the
cytosolic face of the
budding vesicle, thereby
helping the vesicle to pinch
off from the donor (parent)
organelle
Components that participate in budding of coated vesicles
GTP
GDP
5th (final) step:
Small GTP-binding proteins hydrolyze
their binding GTP and pinch off the vesicle
Three types of coated vesicles transport
proteins from organelle to organelle
Vesicle Transport step
COPI Vesicular transport between Golgi cisternae
Golgi ER
COPII ER Golgi
Clathrin Golgi Lysosome
Fusion of all three types of transport vesicles with their
target membranes exhibits several common features:
(1) fusion occurs only after the coats have depolymeraized
protein budding
coat
coated uncoating uncoated
vesicle vesicle
fusion with target membrane
target membrane
Fusion of all three types of transport vesicles with their
target membranes exhibits several common features:
(2) the transport vesicle must specifically recognize the
correct transport membrane
(3) the vesicle and target membranes must fuse, thereby
delivering the contents of the vesicle to the target organelle
Fusion of all three types of transport vesicles with their
target membranes involves a conserved set of proteins:
NSF = N-ethylmaleimide sensitive factor, ATPase
SNAPs = soluble NSF attachment proteins
v-SNARE = SNAP receptor on the transport vesicle
t-SNARE = SNAP receptor on the target membrane
Rab proteins = a family of small GTP-binding proteins
Targeting and fusion of transport vesicles with their target membranes
vesicle
Rab - GTP Rab - GTP
v-SNARE
target t-SNARE
membrane
1st step – Docking:
The attachment of a transport
vesicle to its target membrane
is mediated by binding between
vesicle v-SNAREs and t-SNAREs on
Rab - GTP Rab - GTP
the vesicle and target
membrane, respectively
Rab GTP-binding proteins are
required to facilitate formation
of v-SNARE/t-SNARE
complexes
Targeting and fusion of transport vesicles with their target membranes
vesicle
Rab - GTP Rab - GTP 2nd step – Fusion:
Fusion of the two membranes
requires GTP hydrolysis by
small Rab proteins
Rab - GDP
The NSF/SNAP complex is
Rab - GDP
recruited from the cytosol to
the membrane
SNAPs
NSF
Targeting and fusion of transport vesicles with their target membranes
Rab - GDP Rab - GDP 3rd step – Disassembly of
ATP SNARE complexes:
ADP The NSF/SNAP complex uses
the energy of ATP hydrolysis
by NSF to disassemble the v-
SNARE/t-SNARE complex,
thereby allowing the SNAREs to
be reutilized for subsequent
rounds of vesicle transport
