Transcript lecture 10

10-1
Protein transport and translocation
Protein translocation in bacteria, eukaryotes
- targeting signals
- import, export systems: bacterial, ER, chloroplasts, peroxisomes,
mitrochondria
- nuclear import
Overview of protein transport
and translocation
 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 translocationcompetent 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
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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)
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export signals
Targeting signals
 blue is hydrophilic (H-phil)
 red is hydrophobic (H-phob)
H-phobobic H-philic
 curling lines are helical
 zig-zags are turns
H-phob
import signals
H-phil
H-phob
 ‘OH’ denotes hydroxylated
residues
 ‘+’ denotes positively charged aa’s
 most signals are at the N-terminus
 can be cryptic
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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
 archaea lack SecB, have SRP/FtsY but no
SecA; what drives translocation?
 archaeal SRP, FtsY, SecYEG more closely
related to eukaryotic proteins (SecYEG)
 SecB binds to nascent
chains containing a signal
sequence and maintains the
preprotein in translocationcompetent form, then binds
SecA; SRP docks with
membrane receptor, FtsY
(simpler homologues of
eukaryotic SRP and SRP
receptor)
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
SecB
has an
unexpected
structural
similarity to the
PTB domain
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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
 a post-translational translocation pathway
that makes use of Sec61 also exists;
preproteins are maintained in a translocationcompetent form by Hsp70/Hsp40
 SRP pathway is cotranslational; 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
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Folding in the Endoplasmic reticulum
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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 newlyimported protein
 import into thylakoids (used for
respiration) uses the SRP pathway
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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
Other transport mechanisms likely involve
folded proteins, including the twin-arginine (Tat)
transport system of bacteria, and the cytoplasmto-vacuole targeting pathway of yeast
 various protein oligomers are
imported into peroxisomes
 antibodies with PTS, and 9 nm
gold particles could be imported
Translocation into mitochondria
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 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 systemmediated import of proteins
 protein folding following import
depends on Hsp70, chaperonin (Hsp60)
Import into the nucleus
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 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
Structure of the nuclear pore complex
- RanGTP
bar,
50 nm
+ RanGTP
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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, 99-108.
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, 99-110.
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