Protein Targeting and DegradationIntroduction

Stages of protein synthesisStage 1: activation of amino acidsStage 2: initiationStage 3: elongationStage 4: terminationStage 5: folding and post-translation processing

Translation (T/L)-- The overall process of mRNA-guided protein synthesis (stages 2-4)

Protein synthesis can account for up to 90% of the chemical energy used by a cell for all biosynthetic reaction.

In E. coli-- Molecules used for protein synthesis（總量）: 20000 ribosomes,

100000 related protein factors and enzymes, and 200000 tRNAs (> 35% of the bacterium’s dry weight).

In Eukaryotes-- Almost 300 different macromolecules （種類）cooperate to

synthesize polypeptide.

References = Chap. 27 of Lehninger: Principles of Biochemistry

1. Protein targeting

-- The eukaryotic cell is made up of many structures, compartments, and organelles. Almost all proteins are synthesized in the cytosol. How do proteins destine to their proper location ?e.g., Proteins destined for secretion, integration in the plasma 講義p.2-6

membrane, or inclusion in lysosomes=> modification begins in the endoplasmic reticulum (ER).

e.g., Proteins destined for mitochondria, chloroplasts, or the 講義p.6-9nucleus use three separate mechanisms.

e.g., Proteins destined for the cytosol e.g., import protein by endocytosis=> remain where they are synthesized.

-- Signal sequence, It directs a protein to its appropriate location and is often removed during transport or after the protein has reached its final destinations.e.g., for proteins to mito, chloro, ER => at amino terminus of protein

for proteins to nucleus => embedded in the protein

Protein Targeting and Degradation講義p.13-15

講義p.10-11

F. 1

(1) Post-T/L modification of many eukaryotic proteins (destined for secretion, integration in the plasma membrane, or inclusion in lysosomes) begins in the endoplasmic reticulum.

A. Signal sequences(a) At N-terminus of the protein(b) Composition

--10-15 hydrophobic amino acids--positive charged amino acid(s) at N-terminus--polar amino acids and short-side-chain aa (Ala) near the cleavage site

B. Beginning of post-T/L modificationT/L beginsSRP (signal recognition particle) binds to signal sequence as it emergesSRP binds GTP and ribosome and halts T/LGTP-bound SRP directs ribosome to be bound by SRP receptor on ERSRP recycled as GTP hydrolysisT/L resumes, coupled to translocation of the polypeptide chain into ER through the peptide translocation complexSignal sequence is cleaved by a signal peptidase within the ER lumen upon T/L terminationRibosome dissociated and mRNA detached

F. 2

F. 3

Blue: “+” charge amino acidsYellow: hydrophobic amino acidsBlack/white: polar amino acids

: short-side-chain aa13-16 amino acids in length

Cleavage after the protein is transported into ER

Eukaryotic ER proteinF. 2

Ala

SRP (~70 aa) halts T/L

T/L resumes

SRP releases

Free ribosome

Ribosome attaches to ER membrane

STOP

Cleavage of protein after completion of T/L by signal peptidase inside ERF. 3

C. Glycosylation plays a key role in protein targeting-- A 14 residue core oligosaccharide is built up in a stepwise fashion.

dolichol-phosphate receives oligosaccharide from various nucleotide-conjugated sugar (e.g., UDP-GlcNac, GDP-Man)[This process is inhibited by tunicamycin.]Translocation of dolichol-pyrophosphate with incomplete oligosaccharide across the ER membraneCompletion of core oligosaccharide inside the ER onto dolichol-pyrophosphateThe core oligosaccharide is transferred from dolichol-pyrophosphate to an Asn residue of the protein within ER => N-linked oligosaccharide (The transferase is on the lumenal face of the ER)The released dolichol-pyrophosphate is translocated to the cytosolic phase of ERA phosphate is removed to regenerate dolichol-phosphate

F. 4

No glycosylation=> Inhibit protein targeting

(Unknown mechanism)

14 residue core oligosaccharide

Transferase (inside ER)

Translocation(Unknown mechanism)

N-link

保留 pentasaccharide core (經一連串modification in ER or Golgi)

細節見F.3

F. 4

D. Sorting processing in the Golgi complexProteins are moved from ER to the Golgi complex in transport vesicles (from Cis side to Trans side)O-linked oligosaccharides are added, N-linked oligosaccharides are further modifiedProteins are sorted in the Golgi (on the Trans side) with unknown mechanism

e.g., hydrolases destined for transport to lysosomesN-Acetylglucosamine phosphotransferase recognizes some as yet

unidentified structural of hydrolases and phosphorylates certainmannose residues in the N-linked glycoprotein A receptor protein in the membrane of Golgi recognizes the mannose-6-phosphate signal and binds the hydrolaseVesicles containing the receptor-hydrolase complexes bud from Golgi and make their ways to sorting vesicles(a) low PH and (b) phosphatase-catalyzed removal of phosphate groups from the mannose-6-phosphate=> the receptor-hydrolase complex dissociatedThe receptor is recycled to GolgiThe hydrolases bud from the sorting vesicles and move to lysosome

F. 6

F. 5

Golgi complex

F. 5

N-link

認知 target protein (hydrolase) 之3D結構中之signal patch區域(unknown structure)

Mannose-6-phosphate為signal for targeting to lysosome(在Golgi中有一receptor protein可認知此singal = M-6-P)Budding 過程中因lysosome中:A. PH低B. Ptase切 P* from M-6-P使receptor protein與hydrolase分離=> Hydrolyase moves to lysosome

F. 6

(2) Proteins are targeted to mitochondria and chloroplasts by similar pathways.

A. Signal sequences (a) At N-terminus of the protein (b)Composition

F. 7

B. Process-- begin after a precursor protein has been

completely synthesized The precursor protein bound by cytosolic chaperone ( see F.9(a)(b) )Delivered to receptors (Tom) on the exterior surface of the target organelleTo the protein channel (Tom+Tim) spanning the inner and outer membranes of the organelleTranslocation with the help of (a) ATPase of mHsp70 (in mito.)(b) Electrochemical potential differencesSignal peptide cleaved by proteaseProtein folded by mHsp70 (in mito.)

C. Stop signals-- A signal recognized during transport of the mito

precursor protein through membrane channel.(a) Mito membrane bound protein-- Translocation is halted at a point when a

hydrophobic amino acids span either in outer or inner mito membrane.

(b) Mito soluble intermembrane protein-- Some proteins may re-export to intermembrane

space.

F. 10

F. 8

E.coli chaperonDnaK & DnaJ

= Eukaryotic chaperonHsp70 & Hsp40

F. 9(a)

A

CB

SeeF.9(b)

F. 9(b)Folding mechanism

unknown

membrane channel(Tom+Tim)

Chaperone (F.9)

ATPase

(By additional chaperones e.g., mHsp70)

F. 10

(3) Signal sequences for nuclear transport are not cleaved.A. Examples-- Ribosomal proteins synthesized on

cytosolic ribosomes are imported into thenucleus and assembled into 40S and 60S ribosomal subunits

-- RNAP, DNAP, histones, topoisomerase,cyclins are imported into the nucleus

B. Signal sequences – nuclear localization sequence, NLS) (a) Embedded in protein (b) Composition(c) Is not removed during targetingB. Beginning of post-T/L modification

A protein with an NLS is bound by a complex of importins α and βThe resulting complex binds to a nuclear poreTranslocation is mediated by the Ran GTPaseInside the nucleus, the importin β dissociates from importin αImportin α then dissociates from the nuclear proteinImportins α and β are transported out of the nucleus and recycled

F. 12

F. 11

Soluble receptors in cytosol

F. 12

Also see F. 11-37 RanGTP+

RanGDPRan GAP

RCC

(4) Cells import proteins by receptor-mediated endocytosisA. Examples--low-density lipoprotein (LDL), peptide hormones, ion-carrying protein

transferrin, and circulating proteins destined for degradation

B. ProcessThe proteins bind to receptors in invaginations of the plasma membrane called coated pits-- concentrate cell-surface receptors (on cytosolic side of cell

membrane) for endocytosis-- compose of clathrin, which forms closed polyhedral structuresAs more of the receptor coated pits are occupied by target proteins, the clathrin lattice buds off the membrane to form endocytic vesicleand enter the cytoplasmThe endocytic vesicle fuses with an endosomeThe activity of ATPase in the endosomal membranes reduces the pH, facilitating dissociation of the target proteins with receptorsThe fate of receptor and target protein(a) both recycled, e.g., transferrin(b) both degraded, e.g., hormones, growth factors(c) protein degraded and receptor recycled, e.g., LDL ( see F.14 )

F. 13

Clathrin蛋白(形成特殊三腳結構)

一個 coated pits(由許多clathrin組成的多面體格子狀結構)

F. 13

LDL degraded

LDL receptor recycled

F. 14

Bacteria also use signal sequences for protein targeting-- Bacteria can target proteins to their inner or outer membranes, to the

periplasmic space between membranes, or to the extracellular medium.

-- After T/L, a protein to be exported may fold slowly (The signalsequence impeding the folding.)

A. Signal sequences(a) At N-terminus of the protein (b) CompositionB. Process

Protein binds to cytosolic chaperone protein SecBSecB delivers the protein to SecA, a protein associated with the translocation complex (SecYEG)SecB is released, and SecA inserts itself into the membrane, forcing about 20 amino acids of the protein to be exported through the translocation complexHydrolysis of an ATP by SecA causes its conformation change and withdraw from the membraneRepeats of the above two steps to transport the protein 20 amino acids at a timeFinish the process as the entire protein has passed through

-- Driving force for translocation(a) ATPase activity of SecA(b) Electrochemical potential difference across the membrane

F. 15

F. 16

Blue: “+” charge amino acidsYellow: hydrophobic amino acidsBlack/white: polar amino acids

Cleavage after the protein is transported

: short-side-chain aaBacterial proteinsF. 15

Incomplete folded protein

Cytosolic chaperon

A.ReceptorB.Transloating

ATPase

Electrochemical potential(mechanism unknown)

Translocation complex(位於cytoplasmic membrane)

SecA inserts to membrane

SecA withdraws from membraneafter ATP hydrolysis

repeat 20 aa / time直到 protein 全部送出

F. 16

2. Protein degradation(1) Introduction-- is mediated by specialized system in all cells

(a) ATP-dependent cytosolic system—rapidly degraded proteins(b) lysosome system (in vertebrates)—long half-life proteins

-- The half-lives of eukaryotic proteins vary from 30s to many days.-- Rapidly degraded proteins include:

(a) defective polypeptides during T/L mistakes(b) damaged proteins during normal functioning(c) key regulatory proteins in metabolic pathway

-- The identity of the first residue that remains after removal of the amino-terminal Met residue, and any other post-T/L proteolytic processing of the amino-terminal end, has a profound influence on half-life.* some protein called DBRP (destruction box recognizing protein) can recognize a run of amino acids called destruction box in the amino-terminal of protein to be degraded. (見下段 e.g., cyclin degradation)

T. 1

1

(2) Degradation process in E. coli-- Many proteins are degraded by an ATP-dependent protease call Lon.

(2 ATP are hydrolyzed for every peptide bond cleaved with unknown mechanism)

-- Once a protein is reduced to a smaller size, other ATP-independent proteases complete the degradation process.

(3) Degradation process in Eukaryotes-- ATP-dependent pathway contains proteins called ubiquitin (Ub).

E1: Ub-activating enzymeE2: Ub-conjugating enzymeE3: Ub-protein ligase(E2 and E3 exhibit different specificities for target proteins and thus regulate different cellular processes.)

-- How ubiquitination targets proteins for proteolysis is not yet known.-- After ubiquitination, a large complex called proteasome degrades the

Ub-labeled protein by ATP-dependent processes.-- example: cyclin degradation during cell cycle regulation

F. 17

F. 18

E1: Ub-activating enzymeE2: Ub-conjugating enzymeE3: Ub-protein ligase

一個protein可以被ubiquitination多次

at 多處poly-Ub vs. mono-Ub

F. 17

=E3 ligase (APC)

F. 18

Cdc25

15161

Cdc2

Cyclin B

Also see F.13-8, F.13-9, F.13-13