1. Protein transport and translocation
10-1
Protein transport and translocation
Protein translocation in bacteria, eukaryotes
targeting signals
import, export systems: bacterial, ER, chloroplasts, peroxisomes, mitrochondria
nuclear import

2. Overview of protein transport and translocation
10-2
at least 40% of all cellular proteins are:
inserted into a membrane
translocated into an organelle, nucleus
exported outside the cell or to the periplasm
proteins must be kept in translocation-competent form (i.e., either partially or entirely unfolded
exception is peroxisomes, nucleus
proteins must be folded/assembled after translocation; molecular chaperones are usually involved
translocation is an energy dependent process

3. Protein translocation systems
10-3
Protein translocation systems e e i i i e
IM, inner membrane
IMS, inner membrane space
P, periplasm
OM, outer membrane
TL, thylakoid lumen
TM, thylakoid membrane
SecYEG, Sec61, TOM, TIM, TOC are protein
subunits of the translocation systems
adapted from Schatz and Dobberstein,
Science 271, 1519 (1996)

4. Targeting signals blue is hydrophilic (H-phil)
10-4
Targeting signals
export signals
blue is hydrophilic (H-phil)
red is hydrophobic (H-phob)
curling lines are helical
zig-zags are turns
‘OH’ denotes hydroxylated residues
‘+’ denotes positively charged aa’s
most signals are at the N-terminus
can be cryptic
H-phobobic
H-philic
H-phob
H-phil
H-phob
import signals

5. Translocation in bacteria
10-5
Translocation in bacteria
two major pathways for translocation in bacteria: Sec and SRP pathways
both converge at SecYEG translocon and use SecA, a peripherally-bound ATPase that supplies the energy for translocation
SecB binds to nascent chains containing a signal sequence and maintains the preprotein in translocation-competent form, then binds SecA; SRP docks with membrane receptor, FtsY (simpler homologues of eukaryotic SRP and SRP receptor)
archaea lack SecB, have SRP/FtsY but no SecA; what drives translocation?
archaeal SRP, FtsY, SecYEG more closely related to eukaryotic proteins (SecYEG)

6. Structure and function of SecB
10-6
SecB monomer; functional
complex assembles as a
tetramer (dimer of dimers)
Conserved residues shown to be important for the interaction of SecB with SecA:
Asp27, Glu31, Glu86 (green)
Ile 84
(yellow)
shading of the hydrophobic subsites 1 and 2 in the assembled tetramer; the opposite surface contains the same groove with two separate subsites 1 and 2
PTB (phosphotyrosine Binding) domain
SecB
monomer
has an unexpected structural similarity to the PTB domain

7. Translocation into the ER
10-7
Translocation into the ER
Sec61 is a hetero-trimeric complex composed of a, b, g subunits related to SecYEG
SRP is a ribonucleoprotein complex composed of 7S RNA and numerous proteins
binding of signal sequence is modulated by NAC
SRP pathway is co-translational; SRP mediates arrest of elongation until it docks with SRP receptor; translocation then proceeds through Sec61
SRP is the major pathway used for import into ER
a post-translational translocation pathway that makes use of Sec61 also exists; preproteins are maintained in a translocation-competent form by Hsp70/Hsp40

8. Folding in the Endoplasmic reticulum
10-9
Folding in the Endoplasmic reticulum

9. Translocation into chloroplasts
10-9
Translocation into chloroplasts
Toc components, mediate translocation (Toc75 is the translocon); it is unclear how preproteins are targeted to the channel; Hsp70/Hsp40 may be involved
Hsp70 in both the IMS and the stroma assist the threading of the preprotein into the chloroplast
an Hsp100 chaperone also called ClpC (AAA ATPase) also binds preproteins in the stroma
Hsp70/chaperonin (Cpn60) may assist folding/assembly of newly-imported protein
import into thylakoids (used for respiration) uses the SRP pathway

10. Translocation into peroxisomes
10-10
Translocation into peroxisomes
targeting of proteins is initiated post-translationally by Pex5/7 proteins, which bind the peroxisomal targeting signal (PTS)
translocon not well defined; possibility of vesicular budding?
gated pore that is regulated by membrane proteins?
first organelle demonstrated to import proteins without a PTS, by virtue of assembly with other proteins that contained a PTS
various protein oligomers are imported into peroxisomes
antibodies with PTS, and 9 nm gold particles could be imported
Other transport mechanisms likely involve folded proteins, including the twin-arginine (Tat) transport system of bacteria, and the cytoplasm-to-vacuole targeting pathway of yeast

11. Translocation into mitochondria
10-11
Translocation into mitochondria
delivery of preproteins to mitochondria depends on either Hsp70/Hsp40 or MSF, mitochondrial import stimulation factor (MSF)
evidence now that Hsp90 is also involved
mtHsp70/Tim44/Mge (GrpE) is required for import; Tim44 contains J domain
Big debate:
brownian ratchet or pulling model for Hsp70 system-mediated import of proteins
protein folding following import depends on Hsp70, chaperonin (Hsp60)

12. Import into the nucleus
10-12
Import into the nucleus
nuclear localization signal (NLS) is typically highly basic; e.g., the SV40 large tumor antigen (T ag) has the sequence PKKKRKV
a/b1 importin hetero-dimer recognizes and binds the NLS (or b importin alone)
b importin docks with NPC and mediates interaction with Ran (GDP form)
directionality conferred by nature of guanine nucleotide bound to Ran
Ran binding protein (RanBP) is required for b importin binding to RanGDP; Ran GTPase activating protein (RanGAP) and nucleotide-exchange factor (RCC) are cytoplasmic and nuclear
cytopl. RanGDP required for import; nuclear RanGTP required for release
conversely, RanGTP binds substrate with NES in the export direction
proteins to be imported can be in a native/near native form

13. Structure of the nuclear pore complex
- RanGTP
bar,
50 nm
+ RanGTP

14. Mechanism of import into nucleus
10-14
Mechanism of import into nucleus
some nuclear pore proteins (nucleoporins) contain core FxFG repeats (yellow)
b importin contains ‘heat’ repeats that bind the FxFG repeats (Heat repeats 5, 6, 7 are shown in red, green and blue)
the FxFG repeats interdigitate in grooves formed by the Heat repeats
interaction of b importin with nucleoporins allows transport across the nuclear pore complex
Core FxFG repeats found in nucleoporins.
Each repeat is separated by a ‘linker’ region:
Bayliss et al. (2000) Cell 102,

15. Heat repeat-containing protein
10-15
Heat repeat-containing protein
15 heat repeats of protein phosphatase 2A
conservation is to one side of the repeat structure
Groves et al. (1999) Cell 96,