Translocation: transmembrane protein transport

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Translocation transmembrane protein transport
Protein function requires targeting Approx. 50
proteins transit membranes 30 are membrane
proteins 20 are soluble
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17.0 Overview of sorting of nuclear-encoded
proteins in eukaryotic cells
Figure 17-1
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17.1 Specific uptake-targeting sequences in newly
made proteins are recognized by different
organelles
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Common Principles (cis-gttrans)
N-term signals Partly unfolded precursors Cytosoli
c targeting factors Membrane receptors Hetero-olig
omeric channel Channel gating (both across
within) Peripheral translocation motor NTP
hydrolysis Chaperone-mediated folding
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Translocation into the ER Start w/ consideration
of topology lumenextracellular evo orgin Major
function protein processing for
secretion folding modifications assembly
quality control Other functions calcium
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17.3 The rough ER is an extensive interconnected
series of flattened sacs
Figure 17-11
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17.3 Secretory proteins are found in the ER lumen
immediately after synthesis
Figure 17-12
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17.4 A signal sequence on nascent secretory
proteins targets them to the ER and then is
cleaved off
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17.4 The cotranslational import of proteins into
the ER is studied with microsomes
Figure 17-15
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• Salt wash of microsomes blocks translocation
• Add back after dialysis restores activity
• Single particle purified-gtSRP

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17.4 Structure of the signal recognition particle
(SRP)
Figure 17-17
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• mRNA
• Retic-lysate w/ATP etc.
• SRP

Protease-treated, salt-washed microsomes (very
low protease

Protease-releasate
-gt purify for SRP-receptor alpha
Use alpha to ID beta
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17.4 Two proteins initiate the interaction of
signal sequences with the ER membrane
Figure 17-16
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17.4 Polypeptides move through the translocon
into the ER lumen
Figure 17-18
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Sec61 Complex Alpha 10 TMD Beta, gamma Binds
ribosomes Forms pore (2 nm) 3-4 fold
subunits Gating domain _at_ Cyto side Bip closes
lumen W/ TRAM SRPR Gives translocation
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17.4 GTP hydrolysis powers protein transport into
the ER in mammalian cells
Figure 17-20
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Force is also (!) provided by Bip
Open ATP state
Closed ADP polypeptide state
Rapidly cycles by ATP hydrolysis and exchange
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17.5 Topologies of some integral membrane
proteins synthesized on the rough ER
Figure 17-21
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17.5 Most nominal cytosolic transmembrane
proteins have an N-terminal signal sequence and
an internal topogenic sequence
Figure 17-22
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17.5 A single internal topogenic sequence directs
insertion of some single-pass transmembrane
proteins
Figure 17-23
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17.5 Multipass transmembrane proteins have
multiple topogenic sequences
Figure 17-24
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