Gene Expression: II. Protein Synthesis and Sortingclasspages.warnerpacific.edu/BDupriest/BIO 330/Ch... · Targeting polypeptides to mitochondria and chloroplasts •Given the complexity

© 2012 Pearson Education, Inc.

Lectures by

Kathleen Fitzpatrick Simon Fraser University

Chapter 22

Gene Expression:

II. Protein

Synthesis and

Sorting


How are proteins made?

Where are proteins made?


Figure 22-13A


Figure 22-13B


Figure 22-16, Step 1




Figure 22-16, Steps 3–5




Figure 22-17A


Figure 22-17B


How do proteins get inside

organelles other than those of the

endomembrane system?


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


How does a protein “know” where to go?


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


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


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


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


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


Figure 22-18


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


Figure 22-19


How do proteins stay unfolded?


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


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)


Figure 22-20


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)


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


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


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

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Gene Expression: II. Protein Synthesis and Sortingclasspages.warnerpacific.edu/BDupriest/BIO 330/Ch... · Targeting polypeptides to mitochondria and chloroplasts •Given the complexity