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Directing eukaryotic proteins with signals to ER

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Translocation to ER directed by amino-terminal sequence

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1. The targeting pathway begins with initiation of proteinsynthesis on free ribosomes.

2. The signal sequence appears early in the syntheticprocess, because it is at the amino terminus, which as wehave seen is synthesized first.

3. As it emerges from the ribosome, the signal sequence and the ribosome itself are bound by the large signalrecognition particle (SRP); SRP then binds GTP and haltselongation of the polypeptide when it is about 70 amino acidslong and the signal sequence has completely emerged fromthe ribosome.

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7 The signal sequence is removed by a signal peptidase within the ERlumen;

8 the ribosome dissociates and is recycled.

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Pro tein t arget ing to th e mi toch on dr ion

In the mitochondrion translocation of precursor proteins is anenergy-dependent process requiring ATP utilization.

For small proteins (

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Pro tein t arget ing to th e mi toch on dr ion

Translocation involves specific complexes in the membrane:

Tom (translocation outer membrane)and

Tim (translocation inner membrane)

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First step: precursor protein binding to a translocase systemconsisting of at least nine integral membrane proteins

Tom20, Tom22 and Tom70 are the principal proteins involved inimport.

Tom20 has a single transmembrane domain at the N-terminus with alarge cytosolic domain recognizing precursor proteins from their

targeting signals.

Tom20 cooperates with Tom22 a protein with an exposed N terminaldomain that also binds targeting sequences.

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Tom70, the other major receptor has a large cytosolic domainpreferentially interacting with preproteins carrying internaltargeting information.

Specific recognition by Tom 20, 22, and 70 leads to a second stepinvolving pre-sequence insertion into the Tom40 hydrophilic channel.

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TIM22 binds preproteins exclusively bound for the innermitochondrial membrane.

Tim44 is a peripheral protein that interacts with mitochondrial

chaperones to regulate protein folding and prevent aggregation.

Closely coupled to translocation across the inner membrane isremoval of the N-terminal signal sequence by mitochondrialprocessing proteases (MPP) located in the matrix

The import pathway will target proteins into the matrix ofmitochondria but to reach other destinations the basic importpathway is supplemented by additional sorting reactions.

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Target ing of pro teins to thech lorop las t Targeting sequences contain significant numbers of basic residues, a

high content of serine and threonine residues and are highly variablein length ranging from 20 to 120 residues.

Translocation systems: Tic and Toc

Two proteins, Toc159 and Toc75, act as receptors and form aconducting channel - proteins to traverse outer membranes in an

extended conformation.

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Protein Targeting to Choloroplast Targeting sequences contain

significant numbers of basicresidues, a high content of serineand threonine residues and arehighly variable in length rangingfrom 20 to 120 residues.

Two proteins, Toc159 and Toc75,act as receptors and form aconducting channel with adiameter of 0.8 0.9 nm thatrequires proteins to traverseouter membranes in an extendedconformation.

The Tic proteins involved in

precursor import are Tic110,Tic55, Tic40, Tic22 and Tic20

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Stromal proteins such as ribulose bisphosphate carboxylase,ferredoxin or ferredoxin-NADP reductase do not require additionalsorting pathways.

They are synthesizedwith a single signal sequence that is cleaved by astromal processing peptidase.

Thylakoid proteins such as the light-harvesting chlorophyllcomplexes, reaction centres, and soluble proteins such asplastocyanin must utilize additional targeting pathways.

Targeting is via a bipartite N-terminal signal sequence where the first

portion ensures transfer into the stromal compartment whilst asecond part directs the peptide to the thylakoid membrane wherefurther proteolysis in the lumen removes the second signalsequence.

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Nuclear Transport

Requires the movement of macromolecules throughnuclear pores.

Ribosomal proteins synthesized on cytosolicribosomes are imported into the nucleus andassembled into 60S and 40S ribosomal subunits inthe nucleolus completed subunits are thenexported back to the cytosol.

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Nuclear Transport the nuclear envelope breaks down at each cell division and is re-

established, the dispersed nuclear proteins must b re-imported.

To allow this repeated nuclear importation, the signal sequence that

targets a protein to the nucleus the nuclear localization sequence, NLSis not removed after the protein arrives at its destination.

NLS : consist of four to eight amino acid residues and include severalconsecutive basic (Arg or Lys) residues.

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The complex of the NLS bearing protein and theimportin docks at a nuclear pore and is translocatedthrough the pore by an energy dependent

mechanism that requires the Ran GTPase.

The two importin subunits separate during thetranslocation and the NLS-bearing protein

dissociates from importin inside the nucleus

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PROTEIN TURNOVER

Protein turnover is the balance between proteinsynthesis and protein degradation.

Protein degradation prevents the build up of abnormal and permitsthe recycling of amino acids.

Rapidly degraded proteins include those that are defective orbecause of damage accumulated during normal functioning.

And enzymes that act at key regulatory points in metabolic pathwaysoften turn over rapidly.

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PROTEIN DEGRADATION

In E. coli, many proteins are degraded by an ATP dependent proteasecalled Lon .

protease is activated in the presence of defective proteins or thoseslated for rapid turnover.

two ATP molecules are hydrolyzed for every peptide bond cleaved.

Once a protein has been reduced to small inactive peptides, otherATP-independent proteases complete the degradation process.

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Ubiquitinated proteins are degraded by a large complex known as the 26S proteasome . The proteasome consists of two copies each of at least 32 different

subunits. The proteasome contains two main types of subcomplexes, a

barrellike core particle and regulatory particles on either end of thebarrel.

The 20S core particle consists of four rings; the outer rings areformed from seven subunits, and the inner rings from sevensubunits.

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Three of the seven subunits in each ring have protease activities,each with different substrate specificities.

The stacked rings of the core particle form the barrel-like structurewithin which target proteins are degraded.

The 19S regulatory particle on each end of the core particle contains18 subunits, including some that recognize and bind to ubiquitinatedproteins.

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