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Blueprint Series

Complete Solution
Chapter 13

13
MOVING PROTEINS INTO
MEMBRANES AND ORGANELLES

REVIEW THE CONCEPTS
1
...
In the absence of ER membranes, the entire protein is translated and the ER signal
sequence remains on the protein
...
When translation occurs in the presence of ER-containing microsomes, the protein
is translated into the lumen of the microsomes
...

c
...

If they do not occur at the same, the protein is not properly imported into the ER
where the signal sequence can be cleaved (although there are some examples of
post-translational translocation)
...
a
...
Please note, however, that as translation is completed a portion of the
newly synthesized protein still resides within the translocon
...

b
...
BiP
is luminal protein of the ER and is a member of the Hsc70 family of molecular chaperones
...
Activated BiP is enzymatically active and cleaves ATP
to ADP plus Pi
...

Sequential binding of BiP-ADP to the nascent chain serves to block any sliding of

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CHAPTER 13: Moving Proteins into Membranes and Organelles

the chain back and forth in the translocon and to ratchet the nascent chain
through the translocon
...
Translocation into the mitochondrial matrix occurs through a bipartite Tom/
Tim complex in which Tom is the outer membrane translocon and Tim is the
inner membrane translocon
...
First, ATP
hydrolysis by a cytosolic Hsc70 chaperone keeps the newly synthesized mitochondrial precursor protein unfolded in the cytosol
...
Matrix Hsc70s interact with Tim44 and hence
may be analogous to the BiP/Sec63 interaction at the ER membrane
...
The inside-negative membrane electric potential may serve to electrophorese the amphipathic matrix-targeting sequence toward the matrix
...
SRP (signal recognition particle) acts as a cycling cytosolic factor for the translocation of ER targeted proteins
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In doing this, SRP initiates ribosome binding to ER membranes and positions the nascent chain proximal to the translocon
...
The unfolded
nascent chain then translocates
...
It acts as a molecular chaperone to keep the post-translationally targeted mitochondrial precursor protein
in an open, extended conformation
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These are Pex5, the soluble receptor protein for matrix proteins containing a C-terminal PTS1 targeting
sequence, and Pex7, the soluble receptor protein for matrix proteins containing
an N-terminal PTS2 targeting sequence
...

4
...
Such segments can be referred to as topogenic
sequences
...
They tend to
be about 20 amino acids long, a length sufficient to span the membrane, and
hydrophobic, an appropriate property for a sequence embedded in the hydrophobic lipid bilayer
...
In brief, amino acid sequences of polypeptides may be scanned for
hydrophobic segments of about 20 amino acids long
...
Segments exceeding a threshold value are
expected to be topogenic transmembrane segments
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Such sequences alternate within a multipass membrane protein
...

5
...
It is thought
that the timing of glycosylation modifications is one manner in which misfolded
ER proteins are identified
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CHAPTER 13: Moving Proteins into Membranes and Organelles

6
...
The intermediate is flipped from the cytosolic face
of the ER membrane to the luminal face
...
Short forms of the intermediate are on the wrong
side of the membrane to add to nascent polypeptides within the ER lumen
...

7
...
These include signal peptidase, BiP, oligosaccharyl transferase, various
glycosidases, calnexin and calreticulun, protein disulfide isomerase, peptidylprolyl isomerase, and others
...
Protein disulfide isomerase facilitates the making/breaking of disulfide bonds to ensure correct protein folding
...
The others all
directly support the covalent modification of proteins within the ER lumen
...
Each mutation has a different effect
...
Tom22 together with Tom20 act as outer mitochondrial membrane receptor
proteins for N-terminal matrix targeting sequences
...

b
...
Mutation in Tom70 will have no immediate effect on mitochondrial matrix protein import, as Tom70 does not recognize this class of protein
...
Matrix Hsc70 has a role in the folding of matrix proteins
...
Defective matrix Hsc70 should result in
clogging the Tom/Tim translocon complex with incompletely translocated
proteins
...
Retention of the matrix targeting N-terminal signal sequence because of a
defective matrix signal peptidase might well result in defective folding of the
imported protein
...

9
...
Functionally analogous proteins mediate each process
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Energetically, unlike the situation for mitochondria, there
is no need for a membrane electrochemical gradient for import into chloroplasts
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10
...
How many amino acids must span
the Tom/Tim complex to expose the matrix-targeting sequence to the matrixprocessing protease? DHFR in the presence of the drug methotrexate is locked
into a folded state
...
Instead, it is stuck in the Tom/
Tim complex
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Any unfolded N-terminal DHFR sequence must be
included within the ruler
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11
...
Catalase, like
most other peroxisome-localized enzymes, contains a peroxisometargeting sequence (PTS1) consisting of three amino acids, serine-lysineleucine, at its C-terminus
...
The cata- lase-Pex5 heterodimer moves to
the peroxisome membrane, where it interacts with the Pex14 receptor
located on the membrane
...

12
...
Hence, mutations can selectively affect one or the
other
...
One
approach to determining whether the mutant is primarily defective in
insertion/assembly of peroxisomal membrane proteins or matrix proteins
is to use antibodies to ask by microscopy if either class of proteins localize
to “peroxisomal” structures (e
...
, peroxisome ghosts)
...

13
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The nuclear import receptor binds to the NLS on the cargo
molecule and brings it into the nucleus
...
The receptor is thought to interact
with the FG repeats that are commonly found on nuclear pore complex
proteins, moving from one to thenext as it passes through the nuclear
pore
...
Ran-guanine nucleotide–exchange factor (Ran-GEF) must be present in
the nucleus and Ran-GAP must be in the cytoplasm for unidirectional
transport of cargo proteins across the nuclear pore complex
...
During
nuclear export, Ran-GTP picks upcargo proteins in the nucleus and
carries them to the cytoplasm
...
Once translocated back into the nucleus,Ran
needs to be in the GTP-bound state to pick up more cargo
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