gene expression: ii. protein synthesis and sortingclasspages.warnerpacific.edu/bdupriest/bio...
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Lectures by
Kathleen Fitzpatrick Simon Fraser University
Chapter 22
Gene Expression:
II. Protein
Synthesis and
Sorting
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How are proteins made?
Where are proteins made?
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Figure 22-13A
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Figure 22-13B
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Figure 22-16, Step 1
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Figure 22-16, Step 2
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Figure 22-16, Steps 3–5
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Figure 22-16, Step 6
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Figure 22-17A
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Figure 22-17B
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How do proteins get inside
organelles other than those of the
endomembrane system?
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Posttranslational Import Allows Some
Polypeptides to Enter Organelles After
They Have Been Synthesized
• Proteins destined for the nuclear interior,
mitochondrion, chloroplast, or peroxisome are
imported into these organelles after completion of
translation
• These are synthesized on free ribosomes and
released into the cytosol
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How does a protein “know” where to go?
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Nuclear import
• Each protein released to the cytosol has
localization signals specific to the destination
• E.g. import into the nucleus requires nuclear
localization signals that target proteins for
transport through nuclear pores
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Importing Polypeptides into
Mitochondria and Chloroplasts
• Nearly all polypeptides encoded by mitochondrial
or chloroplast genes are subunits of multimeric
proteins with one or more subunits encoded by
nuclear genes
• Most mitochondrial and chloroplast polypeptides
are synthesized on cytoplasmic ribosome, released
into the cytosol, and taken up by the organelle
within a few minutes
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The transit sequence
• The targeting signal for mitochondrial and
chloroplast polypeptides is a transit sequence
• It is located at the N-terminus of the polypeptide
• Once inside the mitochondrion or chloroplast, the
transit sequence is removed by transit peptidase in
the organelle, often before transport is complete
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Transport complexes
• Polypeptide uptake is mediated by transport complexes in the outer and inner mitochondrial and chloroplast membranes
• Mitochondrial transport complexes are called TOM (translocase of the outer mitochondrial membrane) and TIM (translocase of the inner mitochondrial membrane)
• In chloroplasts they are called TOC and TIC
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Transport complexes and polypeptide
uptake
• Uptake is initiated by components of the transport complexes called transit sequence receptors
• Once the transit sequence binds its receptor, the polypeptide is translocated across the outer membrane through a pore in the TOM or TOC
• If the polypeptide is destined for the interior of the organelle, passage through the TIM or TIC quickly follows
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Figure 22-18
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Evidence for contact sites
• Transport to the interior of the organelle presumably occurs at a contact site where outer and inner membranes lie close together
• Evidence for the model comes from EM, which shows sites of close contact, and from cell-free mitochondrial import systems incubated on ice
• Polypeptides midway through transport can be seen
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Figure 22-19
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How do proteins stay unfolded?
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Polypeptides are transported in an
unfolded state
• Polypeptides entering mitochondria and chloroplasts must be unfolded as they cross the membranes
• Polypeptides experimentally maintained in a folded state are unable to move across the membrane
• To maintain an unfolded state, the polypeptides are bound to chaperone proteins
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The role of chaperones
• Hsp70 class chaperones bind a newly forming polypeptide, keeping it loosely folded (1)
• The transit sequence on the polypeptide binds the receptor component of TOM on the outer mitochondrial membrane (2)
• Then the chaperones are released, accompanied by ATP hydrolysis as the polypeptide moves into the mitochondrial matrix (3)
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Figure 22-20
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The role of chaperones (continued)
• When the transit sequence emerges into the matrix,
it is removed by transit peptidase (4)
• As the remainder of the polypeptide enters the
mitochondria, mitochondrial Hsp70 molecules bind
to it temporarily; subsequent release of Hsp70
requires ATP hydrolysis (5)
• In many cases, Hsp60 chaperones bind to the
polypeptide, then, and help it fold fully (6)
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Targeting of Polypeptides to the
Proper Compartments Within
Mitochondria and Chloroplasts
• Proteins to be imported from the cytosol must be
targeted to the correct compartment within the
organelle
• Mitochondria have four compartments: the outer
membrane, intermembrane space, inner
membrane, and matrix
• Chloroplasts have four compartments, too
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Targeting polypeptides to mitochondria
and chloroplasts
• Given the complexity of mitochondria and
chloroplasts, many polypeptides require multiple
signals for correct targeting
• E.g., targeting to the inner or outer mitochondrial
membranes requires signals directing the
polypeptide to the mitochondrion, plus a
hydrophobic sorting signal to send it to the final
destination
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Targeting polypeptides to mitochondria
and chloroplasts
• Multiple signals are also involved in directing
chloroplast polypeptides to their final destinations
• E.g. In the stroma the transit sequence required for
chloroplast targeting is cleaved, unmasking a
hydrophobic thylakoid signal sequence
• It targets the polypeptide for the thylakoid
membrane or lumen