There are three different
types of targeting
systems to determine the
organization of a protein
in a membrane.
Combinations of 3 types.
• Characteristic of a given protein
• Graphic way of identifying hydrophobic regions in the protein that can serve as transmembrane
domains, signal sequences, signal anchor sequencesor stop-transfer sequences.
⇨ Determine average hydropathy index for 5 amino acidwindow
⇨ Move window over one amino acid, determine again
⇨ Plot hydropathy average vs. position of amino acidin polypeptide
Positive amino acid sequences flanking signal sequences help predict protein orientation
⇨ Understanding the characteristics of the polypeptide chain allow you to determine the
orientation of a particular membrane protein (n-terminus in lumen vs in cytosol). Post-translational modifications in the ER
As proteins are being synthesized and enter the ER,they undergo a large number of processes.
(Newly made proteins in the ER membrane and lumen undergo modification)
⇨ Disulfide bond formation
⇨ Assembly into multimeric proteins
⇨ Only properly folded and assembled proteins can exit the ER and pass through Golgi to final
destination. “quality control”
Correct disulfide bond formation
• Form between cysteine residues.
The oxidation of sulfhydryl groups
in successive cysteine residues in a
polypeptide chain results in the
formation of disulfide bridges.
• PDI mediates this process.
• (PDI = Protein disulfide isomerase)
• Oxidized PDI
Protein with cysteines comes into
the ER. Oxydized PDI will react
with successive cysteines creating
a disulfide bridge.
• ERO1 will react with reduced PDI
and convert it back to its oxidized
form, reducing the sulfhydrides
which oxidize spontaneously in the
ER with molecular oxygen.
Some disulfide bonds may need to be rearranged. Incorrectly formed disulfide bonds are
exposed and can react with reduced PDI which will mediate the breaking and reforming o
disulfide bonds to stabilize the proper form of the mature polypeptide. Protein glycosylation in the ER
• Most plasma membrane and secreted proteins containattached carbohydrate chains
• Glycosylation may be N-linked or O-linked
• N-linked: A preformed oligosaccharide is added to Asn (Asparigine)residues of many
proteins in the ER, as they are synthesized
- Added only at Asn residues flanked by X-Ser or X-Thr (C-side)
• O-linked: Attachment of carbohydrate residues (often complex) to syrine or threonine.
• Oligosaccharide: (Glc) 3Man) (9lcNAc) for2ed on Dolichol phosphate, an ER phospholipid.First
step in formation of Dolichol oligosaccharide inhibited by tunicamycin
• Tunicamycin is an antibiotic, a very useful tool to observe the fate of the protein if it is
not successfully modified.)
• Oligosaccharide processed prior to passage to Golgi: quality control Quality control: oligosaccharide modification & chaperone proteins (Calnexin, Calreticulin)
Capable of binding to oligosaccharide
with final glucose.
Unfolded protein response
• BiP = Hsc 70 protein of ER lumen,
facilitates protein folding.
• Unfolded proteins bind to BiP, release
BiP from Ire
• Unbound Ire dimerizes ▯ ac▯vates
• Endonuclease cleaves Hac1 mRNA,
allows production of Hac1 protein
Usually occurs during heat
• Hac1 enters nucleus, stimulates
transcription of folding proteins Mitochondrial protein import
• You can take your protein
and synthesize it in your
necessary for protein
mitochondria needed a
to take up proteins.
Mitochondrial signal sequences
• Most proteins that go to the mitochondria have a sequence that targets them to the matrix.
- Have N-terminal signal sequence that is removed bypeptidase
• Outer membrane, some inner membrane: internal signal sequences
• Signal sequences necessary and sufficient
for targeting. Attaching a signal sequence
to any protein will direct it to the
• Matrix signal sequences: amphipathic helix
Hydrophobic and Hydrophilic regions.
Positive amino acids are on one side of the
helix, and hydrophobic amino acids are on
the other. Hydrophobic AAs attach to a hydrophobic crevice in a receptor on the outer mitochondrial membrane.
Protein import into matrix: chaperones and
• Newly made protein associates with
chaperone (Hsc70). Energy from ATP
hydrolysis keeps protein unfolded.
• Targeting sequence associates with import
• Precursor transferred to Tom 40 import
channel in outer membrane, associates with
Tim channel in inner membrane
• Protein drawn into matrix by proton motive
force, associates with matrix Hsc70
• Targeting sequence cleaved, association with
Targeting to the inner
- Path A and path B both
feature matrix-targeting signal
Interact with a receptor on the
outer membrane. Go from TOM 40
translocon to TIM23-TIM17
Protein either has an internal stop-
transfer sequence which stops
protein transfer at TIM23-17 and the protein is released. C terminus in intermembrane space, n-terminus in the matrix.
Second pathway used by proteins that are often membrane proteins, components or respiratory
pathways. Enters matrix completely and associates with Oxa1. Allows for proper assembly and
A third pathway is used by ATP/ADP translocase andother proteins with multiple transmembrane
domains. They have internal targeting sequences, but never reach TIM23-17, but rather reach another
translocon which allows them to
Inter-membrane space targeting
• Precursors have “bi-
matrix targeting + IMS
• Precursor enters like matrix
protein. Matrix targeting seq
• IMS targeting seq stops in I.M.
during import, dissociates
from Tim channel, IMS
targeting sequence cleaved
• Outer membrane: matrix
targeting but “stop transfer”
sequence anchors in outer
membrane before transfer to
matrix Lecture 17
Overview of major protein transport routes in the secretory pathway
From ER to Golgi: A protein in the ER can be transmitted to a Cis-Golgi
network, and then to the trans-Golgi network through Golgi apparatus.
This is mediated by cisternal progression.
From Golgi to ER: Once it reaches the trans-Golgi network is either
sorted to plasma membrane, or to the lysosome.
What allows soluble and membrane proteins to be moved accurately
and efficiently within the secretory pathway?
Vesicles carry proteins through the system.
Three well-characterized types of transport vesicles that work in different transport steps
- COP2: From ER to Golgi
- COP1: From Golgi to the ER
Vesicles of the same type have a relatively uniqueshape and size
Unique morphology. Each type of vesicles is associated with differentcoat proteins covering their sytosolicsurface
Very small GTP binding
proteins also located on
coat protein surface.
The role of coat proteins
1. Vesicle formation: shape and assemble a vesicle into high
curvature of the membrane that is typical of a transport
vesicle in ≈70nm in diameter
2. Cargo selection: determine which proteins are
incorporated into shaped vesicles
Formation of vesicles is initiated by a family of coat-forming small Arf GTP binding protein
1. Sar1 for COPII vesicle formation in the ER membrane
2. Arf1 for COPI vesicle formation in Golgi
3. Arf6 for Clathrin vesicle formation in trans-Golgi
network or plasma membrane
Arf GTPasec ycles between GDP-
bound (inactive) and GTP-bound
(active) forms How the cell can initiate the formation of a COPIIvesicle.
1. Sec12, a specific Sar1 GEF embedded in the ER, recruits
cytosolic, inactive Sar1-GDP, and switches it into active Sar1-
GTP; once activated, the N-terminal hydrophobic helix of Sar1-
GTP is exposed and inserted into the ER
2. Membrane-bound Sar1-GTP recruits Sec23-Sec24. Sec24
interacts with membrane cargoes and cargo receptorsso cargo
proteins are recruited into forming vesicles