Protein Sorting, Signal sequences, Nuclear transport

1. Protein Sorting and Targeting
Raja Ishaq Nabi Khan
Department of Biotechnology

2. Protein targeting
Protein targeting or protein sorting is the mechanism by which a
cell transports proteins to the appropriate positions in the cell or
outside of it.

3. TARGETING PATHWAYS

4. Targeting signals
Targeting signals are the pieces of information that enable the cellular transport
machinery to correctly position a protein inside or outside the cell.
This information is contained in the polypeptide chain or in the folded protein.
In the absence of targeting signals, a protein will remain in the cytoplasm
• The presequences and
• The internal targeting peptides
There are two types of
targeting peptides.

5. Targeting signals

6. Proteins Can Move Between Compartments in Different Ways
Gated
transport(Nucleus )
Transmembrane
transport(Mitochondria,
Peroxisomes,)
Vesicular transport
(E.R)

7. THE TRANSPORT OF MOLECULES BETWEEN THE
NUCLEUS AND THE CYTOSOL – Gated transport
This transport, for the small proteins (e.g,
histones), seems to take place through nuclear
pores of 70 Å diameter; but for larger proteins
(> 90 kd), a short peptide sequence (or signal
sequence) appears to be necessary.
-Pro-Lys-Lys-Lys-Arg-Lys-Val-
The nuclear localization sequences can also
accelerate the entry of small proteins. The transport
of large proteins into nuclei is powered by ATP
hydrolysis.
None of the nuclear localization
signals are cleaved on entry into the
nucleus.!!!

8. IMPORT AND EXPORT OF PROTEINS TO NUCLEUS
Import
A protein will bind
to importin and, together, the
complex will move through the
nuclear pore.
At this point, Ran-GTP will bind
to the importin-protein complex,
Binding will cause the importin
to lose affinity for the protein.
The protein is released,
The Ran-GTP/importin
complex will move back out of
the nucleus through the
nuclear pore.
Export
The exportin binds the cargo
and Ran-GTP
The complex diffuses through
the pore to the cytoplasm,
In cytoplasm the complex
dissociates.
Cargo release to the cytoplasm
occurs upon GTP hydrolysis
with the help of RanGAP

9. Transmembrane transport

10. Mitochondria TOM transports-mitochondrial
precursor proteins , nucleus-encoded
mitochondrial proteins.
TIM23-proteins into the matrix space.
TIM22-mediates the insertion of a
subclass of inner membrane proteins,
including the carrier protein that
transports ADP, ATP, and phosphate.
OXA-mediates the insertion of inner
membrane proteins .

11. Protein import by mitochondria
Energy required
Energy

12. Protein import from the cytosol into the inner
mitochondrial membrane or intermembrane space

13. Sorting of proteins to peroxisomes
All peroxisomal proteins are encoded by nuclear genes.
To date there are two types of known Peroxisome Targeting
Signals (PTS):
Peroxisome targeting signal 1 (PTS1): a C-terminal tripeptide with a
consensus sequence (S/A/C)-(K/R/H)-(L/A). The most common
PTS1 is serine-lysine-leucine (SKL). Most peroxisomal matrix
proteins possess a PTS1 type signal.
Peroxisome targeting signal 2 (PTS2): a nonapeptide located near the
N-terminus with a consensus sequence (R/K)-(L/V/I)-XXXXX-
(H/Q)-(L/A/F) (where X can be any amino acid).
There are also proteins that possess neither of these signals. Their
transport may be based on a so-called "piggy-back" mechanism:
such proteins associate with PTS1-possessing matrix proteins and
are translocated into the peroxisomal matrix together with them.

14. The signal hypothesis
The Endoplasmic Reticulum

15. SRP direct ribosomes to the ER membrane

17. Protein translocation

18. Memrane proteins
Single-pass transmembrane protein

19. Major topological classes of integral membrane
proteins synthesized on the rough ER

20. Soluble proteins
Protein glycosylation in the rough ER

21. Oligosaccharides are used as tags to mark the state of protein folding
Calnexin and Calireticulin
Manosidase

22. The export and degradation of
misfolded ER proteins

23. GPI ANCHORED PROTEINS
Glycosylphosphatiyl inosital anchored proteins

24. VESICULAR TRANSPORT

25. Transport between organelles is mediated by coated
vesicles
Clathrin coated vesicles mainly involved in endocytosis
COP coated vesicles mediate ER to Golgi and back

26. Processing in Golgi Bodies
Glycosylation of proteins:
N-linked oligosaccharide chains on proteins are altered as the proteins pass through the
Golgi cisternae en route from the ER.
Further modifications of N-linked oligosaccharide in the Golgi apparatus gives two broad
classes of N-liked oligosaccharides, the complex oligosaccharides and the high mannose
oligosaccharides.
Complex oligosaccharides High mannose oligosaccharides.
High mannose oligosaccharides have no new
sugars added to them in the Golgi body. They
contain just two N-acetylglucosamines and
many mannose residues.
Complex oligosaccharides, by contrast,
can contain more than the orginal two
N-acetylglucosamines as well as a
variable number of galactose and sialic
acid residues and, in some cases,
fucose

27. Two secretory pathways; constitutive and regulated
Default pathway
for ER/Golgi
proteins
If no address
label, then
secrete
Signal required to trigger
secretory granule fusion
Example - neurotransmitter
release
Inside lumen is equivalent to
outside of cell secretory_pathway.mov

28. If secretion is default, how are resident ER proteins retained?
C, M, T GolgiE
R
Plasma
membrane
OutsideCGN TGN
Constituitive
secretion
Secretory granule
Regulated
secretion
Ex: BiP is a member of the HSP70 family that functions in the ER…
They aren’t!
BiP escapes from ER and must be “retrieved” from the Golgi…
C-terminal KDEL in BiP sequence functions as retrieval signal…
BiP KDEL
KKXX
KDEL-R
KDEL-receptors in Golgi direct retrieval/recycling…
KKXX at C-terminus of KDEL-R binds COPI coat and targets back to ER…

29. Summary so far of protein targeting, revisited…
Cytoplasm
Secretion/membrane proteins
Secretory
vesicles
RetrievalTransport
(constituitive
secretion)
(regulated
secretion)
Protein
targetingVesicletargeting
RER
Golgi
Plasma membrane
See ECB figure 14-5
Default
Signal sequence
(hydrophobic a-helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
Lysosomes
?
Default
How are proteins targeted to the lysosome?

30. Mannose-6-P targets proteins from Golgi to lysosome
Cis Golgi
Network (CGN)
Trans Golgi
Network (TGN)
RER
M6P receptor recycling back to Golgi
Transport via clathrin-coated vesicles to… Lysosome
M6P receptor in TGN directs transport of enzymes to lysosome via clathrin-coated
vesicles
Addition of M6P to lysosomal enzymes in cis-Golgi
Patients with I-cell disease lack phosphotransferase needed for addition of M-6-P to
lysosomal proteins in fibroblasts… secreted…
Lysosomal
hydrolase
(precursor)
Addition of
M6P
Removal of phosphate &
proteolytic processing…
Mature
hydrolase
M6P receptor
Clathrin
coat
Uncoupling
(pH 5)

31. PROTEIN TARGETING, REVISITED
Cytoplasm
Secretion/membrane proteins
Secretory
vesicles
Lysosomes
RetrievalTransport
(constituitive
secretion)
(regulated
secretion)
Protein
targetingVesicletargeting
RER
Golgi
Plasma membrane
Signal sequence
(hydrophobic a-helix)
KDEL (soluble proteins)
KKXX (membrane proteins)
M6P
Default
or
signal?
Default
or
signal?

32. EUKARYOTIC PROTEIN TRANSPORT ACROSS MEMBRANES
Lipidation
Lipidation is a method to target proteins to membranes in organelles
(endoplasmic reticulum [ER], Golgi apparatus, mitochondria), vesicles
(endosomes, lysosomes) and the plasma membrane. The four types of
lipidation are:
 C-terminal glycosyl phosphatidylinositol (GPI) anchor-
 N-terminal myristoylation
 S-myristoylation
 S-prenylation
Each type of modification gives proteins distinct membrane affinities, although
all types of lipidation increase the hydrophobicity of a protein and thus its
affinity for membranes. The different types of lipidation are also not mutually
exclusive, in that two or more lipids can be attached to a given protein