Protein Structure, Targeting and Sorting

8/8/2019 Protein Structure, Targeting and Sorting

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(a) The linear sequence of

amino acids (10 structure)

folds into helices or sheets

(20 structure) which pack

into a globular or fibrous

domain (30 structure).

Some individual

proteins self-associate into

complexes (40 structure).

(b) Proteins display

functions that arise from

specific binding

interactions and

conformational

changes in the structure of

a properly folded protein.


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Sometimes the primary sequence of amino acids is sufficient tospontaneously direct the folding of proteins into their proper

shape.

However, often newly-made proteins require the help of

molecular chaperones to attain their final shape. Members of the

heatshock protein family (Hsp70 and Hsp60) briefly bind to andstabilize hydrophobic regions of proteins (especially rich in Trp,

Phe, Leu) allowing proper folding instead of aggregation with

other immature proteins.

Heat-denatured proteins can be renatured through the activity

of molecular chaperones and heatshock proteins are made

during times of stress. A number of diseases, including Alzheimer's disease, may be

considered to be protein-folding diseases.

Prion diseases, such as "mad cow" disease, may "self-

propagate" based upon a misfolded protein that can, in turn,

misfold other versions of the same protein.


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(a) Many proteins fold into their

proper 3-D structures with the assistance of Hsp70-like proteins (top). These chaperonestransiently bind to a nascent polypeptide as it emerges from a ribosome. Proper folding of

other proteins (bottom) depends on chaperonins such as the prokaryotic GroEL, a hollow,

barrel-shaped complex of 14 identical 60,000-MW subunits arranged in two stacked rings.

One end of GroEL is transiently blocked by the co-chaperonin GroES, an assembly of

10,000-MW subunits. (b) In the absence of ATP or presence of ADP, GroEL exists in a tight

conformational state that binds partly folded or misfolded proteins. Binding of ATP shifts

GroEL to a more open, relaxed state, which releases the folded protein.


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The ER membrane-bound chaperone protein calnexin, or aresident chaperone calreticulin binds to incompletely folded

proteins, trapping the protein in the ER. Glucosyl transferase

determines whether the protein is folded properly or not: if the

protein is still incompletely folded, the enzyme renews the

protein's affinity for calnexin & retains it in the ER. The cycle

repeats until the protein has folded completely.


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Misfolded soluble proteins in the ER lumen or membrane

proteins are translocated back into the cytosol, where they are

deglycosylated, ubiquitylated, and degraded in proteasomes.

Misfolded proteins are exported through the same type of

translocator that mediated their import; accessory proteins

allow it to operate in the export direction.


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(a) Enzyme E1 is activated byattachment of an ubiquitin (Ub)

molecule (1) and then transfers

this Ub molecule to E2 (2).Ubiquitin ligase (E3) transfers

the bound Ub molecule on E2 to

the side-chain-NH2 of a lysine

residue in a target protein (3).Ub

molecules are added to the

target protein by repeating steps13 , forming a polyubiquitin

chain that directs the tagged

protein to a proteasome (4).

Within this complex, the protein

is cleaved into small peptide

fragments (5).

(b) Computer-generated imagereveals that a proteasome has a

cylindrical structure with a cap

at each end of a core region.

Proteolysis of ubiquitin-tagged

proteins occurs along the inner

wall of the core.


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After the amino chain is made, many

proteins undergo posttranslational

processing (including removal of

stretches of amino acids).

1. In prokaryotes, the N-formylgroup is always removed in the

mature protein and often the

methionine and, sometimes, a

number of N-terminal amino acids

are cleaved away from the final

protein product.

Example: Proinsulin is convertedto the active hormone by the

enzymatic removal of a long

internal section of polypeptide.

The two remaining chains

continue to be covalently

connected by disulfide bonds

connecting cysteine residues in

insulin.

2. Recently discovered, the process

of protein splicing (analagous to

RNA splicing) removes inteins

and splices the exteins together

to make a mature protein.


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Free and bound populations of ribosomes are activeparticipants in protein synthesis.

Free ribosomes are suspended in the cytosol and

synthesize proteins that reside in the cytosol.

Bound ribosomes are attached to the cytosolic side

of the endoplasmic reticulum.

They synthesize proteins of the endomembrane

system as well as proteins secreted from the cell.

Secretory proteins are released entirely into the

cisternal space, but membrane proteins remainpartially embedded in the ER membrane.

While bound and free ribosomes are identical in

structure, their location depends on the signal

peptidase of proteins that they are synthesizing.

PROTEIN TARGETING AND SORTING


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Overview of major protein-sorting

pathways in eukaryotic cells.


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In cotranslational import, proteins to be targeted to the endoplasmic reticulum initiallyhave an N-terminal peptide, the ER signal sequence, translated by a cytosolic ribosome.

The ER signal sequence is bound by a signal-recognition particle (SRP), a

ribonucleoprotein complex composed of 6 peptides and a 300 nucleotide RNA molecule.

The SRP binds to the SRP receptor to dock the ribosome on the ER membrane.

When the SRP receptor binds GTP, the nascent polypeptide enters the pore.


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The SRP is released with hydrolysis of the GTP.

The growing polypeptide translocates through a hydrophilic pore created by one or more

membrane proteins called the translocon.

The ribosome fits tightly across the cytoplasmic side of the pore and the ER-lumen side is

somehow closed off until the polypeptide is about 70 amino acids long.

When the polypepide is complete, the signal peptidase cleave the signal to release the

protein into the ER lumen while retaining the signal peptide, for a time, in the membrane.

Afterwards the ribosome is released and the pore closes completely.


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Other kinds of signal peptides are used to target polypeptides to

mitochondria, chloroplasts, the nucleus, and other organelles that

are not part of the endomembrane system.

In these cases, translation is completed in the cytosol before thepolypeptide is imported into the organelle.

Each of these polypeptides has a postal code that ensures its

delivery to the correct cellular location.

In principle, a signal could be required for either retention in, or

exit from a compartment.


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Major topological classes of integral membrane proteins synthesized on

the rough ER. The hydrophobic segments of the protein chain form helices

embedded in the membrane bilayer; the regions outside the membrane arehydrophilic and fold into various conformations. All type IV proteins have

multiple transmembrane helices. The type IV topology depicted here

corresponds to that of G proteincoupled receptors: seven helices, the N-

terminus on the exoplasmic side of the membrane, and the C-terminus on the

cytosolic side. Other type IV proteins may have a different number of helices

and various orientations of the N-terminus and C-terminus.


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Integral membrane proteins are inserted into the ER

membrane as they are made, rather than into the lumen.


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Posttranslational importallows some polypeptides to enter

organelles after protein synthesis. Like cotranslational import

into the ER, posttranslational import into a mitochondrion (and

chloroplast) involves a signal sequence (called a transit

sequence), a membrane receptor, pore-forming membrane

proteins, and a peptidase.


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In the mitochondrion, the membrane receptor

recognizes the signal sequence directly without

the intervention of a cytosolic SRP.

Furthermore, chaperone proteins play several

crucial roles in the mitochondrial process:

o Chaperones keep the polypeptide partially

unfolded after synthesis in the cytosol so thatbinding of the transit sequence and

translocation can occur.

o Chaperones drive the translocation itself by

binding to and releasing from the polypeptidewithin the matrix, an ATP-requiring process

o Chaperones often help the polypeptide fold

into its final conformation.


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Protein import into the mitochondrial

matrix. Precursor proteinssynthesized on cytosolic ribosomes are

maintained in an unfolded or partially

folded state by bound chaperones,such as Hsc70 (1). After a precursorprotein binds to an import receptor

near a site of contact with the inner

membrane (2), it is transferred into the

general import pore (3). The

translocating protein then moves

through this channel and an adjacent

channel in the inner membrane (4-5).

Note that translocation occurs at rarecontact sites at which the inner and

outer membranes appear to touch.

Binding of the translocating protein by

the matrix chaperone Hsc70 and

subsequent ATP hydrolysis by Hsc70

helps drive import into the matrix.Once the uptake-targeting sequence is

removed by a matrix protease andHsc70 is released from the newly

imported protein (6), it folds into the

mature, active conformation within the

matrix (7). Folding of some proteins

depends on matrix chaperonins.


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Pathways for transporting proteins from the cytosol to the

inner mitochondrial membrane.


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In all three pathways, proteins cross the outer membrane via

the Tom40 general import pore.

Proteins delivered by pathways A and B contain an N-

terminal matrix-targeting sequence that is recognized by the

Tom20/22 import receptor in the outer membrane.

Although both these pathways use the Tim23/17 inner-

membrane channel, they differ in that the entire precursor

protein enters the matrix and then is redirected to the innermembrane in pathway B. Matrix Hsc70 plays a role similar its

role in the import of soluble matrix proteins.

Proteins delivered by pathway C contain internal sequences

that are recognized by the Tom70 import receptor.

A different inner-membrane translocation channel (Tim22/54)is used in this pathway.

Two intermembrane proteins (Tim9 and Tim10) facilitate

transfer between the outer and inner channels.


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Two pathways for transporting

proteins from the cytosol to the

mitochondrial intermembrane

space. Pathway A, the major onefor delivery to the inter-membrane

space, is similar to pathway A fordelivery to the inner membrane.

The major difference is that the

internal targeting sequence in proteinssuch as cytochrome b2 destined for

the intermembrane space isrecognized by an innermembrane

protease, which cleaves the protein on

the inter-membrane-space

side of the membrane. Thereleased protein then folds

and binds to its hemecofactor within the

intermembrane

space. Pathway B

involves directdelivery to the

intermembranespace through the

Tom40 general

import pore in the

outer membrane.


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Two of the four pathways for transporting

proteins from the cytosol to the thylakoid

lumen. In these pathways, unfoldedprecursors are delivered to the stroma via the

same outer-membrane proteins that import

stromal-localized proteins. Cleavage of the N-terminal stromal-import sequence by a

stromal protease then reveals the thylakoid-

targeting sequence. At this point the two

pathways diverge. In the SRP dependent

pathway (left), plastocyanin and similarproteins are kept unfolded in the stromal

space by a set of chaperones and, directed by

the thylakoid targeting sequence, bind to

proteins that are closely related to the

bacterial SRP, SRP receptor, and SecY

translocon, which mediate movement into the

lumen. After the thylakoid-targeting sequence

is removed in the thylakoid lumen by a

separate endoprotease, the protein folds

into its mature conformation. In the pH

dependent pathway (right), metal-binding

proteins fold in the stroma, and complexredox cofactors are added. Two arginine

residues (RR) at the N-terminus of the

thylakoid-targeting sequence and a pH

gradient across the inner membrane are

required for transport of the folded protein

into the thylakoid lumen. The translocon in

the thylakoid membrane is composed of

at least four proteins related to proteins in

the bacterial inner membrane.


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(1) Catalase and most other peroxisomal

matrix proteins contain a C-terminal

PTS1 uptake-targeting sequence (red)

that binds to the cytosolic receptor Pex5.

(2) Pex5 with the bound matrix protein

interacts with the Pex14 receptor located

on the peroxisome membrane. (3) The

matrix proteinPex5 complex is then

transferred to a set of membrane

proteins (Pex10, Pex12, and Pex2) that

are necessary for translocation into the

peroxisomal matrix by an unknown

mechanism. (4) At some point, either

during translocation or in the lumen,

Pex5 dissociates from the matrix

protein and returns to the

cytosol, a process that involves

the Pex2/10/12 complex and

additional membrane and

cytosolic proteins. Note that

folded proteins can be imported

into peroxisomes and that the

targeting sequence is not

removed in the matrix.

Import of

peroxisomalmatrix

proteins

directed by

PTS1

targeting

sequence.


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Mutations are changes in the genetic material

of a cell or virus. MUTATION AND DNA REPAIR MECHANISMS.pptx

These include large-scale mutations in which

long segments of DNA are affected (forexample, translocations, duplications, and

inversions).

A chemical change in just one base pair of a

gene causes a spontaneous or point mutation. If these occur in gametes or cells producing

gametes, they may be transmitted to future

generations.


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For example, sickle-cell disease is caused by a

mutation of a single base pair in the gene that codes

for one of the polypeptides of hemoglobin. A change in a single nucleotide from T to A in the

DNA template leads to an abnormal protein.


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hill.com/olc/dl/120077/bio25.swf


hill.com/olc/dl/120077/micro06.swf


hill.com/olc/dl/120077/bio30.swf

http://www.wiley.com/college/boyer/0470003790/ani

mations/translation/translation.htm

Protein Structure, Targeting and Sorting

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