Posttranslational Modification and Targeting of Proteins · 2/1/2017 · Posttranslational Modification and Targeting of ... specific proteins across a membrane from the cytosol into

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Posttranslational Modification

and Targeting of Proteins

Graduate Biochemistry

Term 2/2016

Assist. Prof. Dr. Panida Khunkaewla

School of Chemistry, Institute of Science

Suranaree University of Technology

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What is posttranslational Modification?

“Modification of nascent protein by adding groups or cleavage

of some parts to get mature protein”

Key wards:

Adding

Deleting

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Diversity of Posttranslational modification of proteins

Cleavage of signal peptides

Phosphorylation

Amidation

Glycosylation

Hydroxylation

Ubiquitination

Addition of prosthetic groups

Iodination

Adenylation

Sulfonation

Prenylation

Myristoylation

Acylation

Acetylation

Methylation

Oxidative crosslinking

N-Glutamyl cyclization

Carboxylation

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Nucleus acetylation, phosphorylation

Lysosome mannose-6-phosphate labelled N-linked sugar

Mitochondria N-formyl acylation

Golgi N- and O-linked ologosaccharide, sulfation,

palmitoylation

ER N-linked oligosaccharide, GPI-anchor

Cytosol acetylation, methylation, phosphorylation,

Ribosome myristoylation

Plasma membrane N- and O-glycosylation, GPI-anchor

Extracelullar fluid N- and O-glycosylation, acetylation,

phosphorylation

Extracellular matrix N- and O-glycosylation, phosphorylation,

hydroxylation

Location Modification

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Proteolytic cleavage

Covalent cleavage of one or more peptide bonds in protein substrates by protease. Schematic

processing of preproinsulin to proinsulin by signal peptidase in the ER and of proinsulin to insulin by

proprotein convertases in the trans Golgi network.

Christopher T. W. Posttranslational Modification of Proteins, 2006, Robert and Company Publisher.

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Covalent addition of a substrate fragment to protein side chain catalyzed by a posttranslational modification

enzyme. Examples shown are generic phosphorylation, acetylation, and O-glycosylation.


Covalent modifications

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Irreversible Modification

Irreversible changes include the proteolysis of peptide bonds, installation of phopsphopantertheinyl groups in

fatty acids synthase, and N-methylation of lysine residues in histones.


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Reversible Modifications

Reversible covalnt modifications including protein phosphorylations (due to balance of kinase and phosphatase),

histone acetylation (due to the balance of histone acetyltransferase and histone deacetylase), and protein

ubiquitylations (due to the balance of ligases and deubiquitylating hydrolase


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Purpose of posttranslational modification.

Targeting of protein

Stability of protein

Function of protein

Control protein activity

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Targeting of proteins

“How are synthesized proteins directed to their final cellular destination?”

Secreted protein, membrane protein, inclusion protein in lysosomes

Protein destines to mitochondria Protein destines to chloroplast

Protein destines to nucleus Cytoplasmic proteins

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Proteins can move between compartments in different ways

Cycles of membrane budding and fusion permit the lumen of any of these organelles to communicate with

any other and with the cell exterior by means of transport vesicles.

Blue arrows indicate the extensive network of outbound and inbound traffic route.

Some organelles, most notably mitochondria and (in plant cells) plastids do not take part in this

communication and are isolated from the traffic between organelles shown here.

Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002.

http://www.garlandscience.com/textbooks/0815341059.asp

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Three major ways of protein moving between different compartments

1. In gated transport, the protein traffic between the cytosol and nucleus occurs through

the nuclear pore complexes. The nuclear pore complexes function as selective gates

that actively transport specific macromolecules and macromolecular assemblies,

although they also allow free diffuse of smaller molecules.

2. In transmembrane transport, membrane-bound protein translocators directly transport

specific proteins across a membrane from the cytosol into a space that is topologically

distinct. The transported protein molecules usually must unfold to snake through the

translocator. The initial transport of selected proteins from the cytosol into the ER lumen

or from the cytosol into mitochondria, for example, occurs in this way.

3. In vesicular transport, membrane-enclosed transport intermediates—which may be

small, spherical transport vesicles or larger, irregularly shaped organelle fragments-ferry

protein from one compartment to another.



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Signal sequence

Signal sequence is a short

sequence of amino acids that directed a

protein to it appropriate location in the

cell and, for many protein, is removed

during transport or after the protein has reached to its final destination.

1970

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Albert B. et. al. Molecular Biology of the Cell, 4th ed, 2000

The Nobel Prize in Physiology or Medicine 1999

"for the discovery that proteins have intrinsic signals that govern their transport and localization in

the cell” PROTEIN ZIP CODES

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Signal sequence directed to ER

The carboxyl terminus of the signal sequence is defined by a cleavage site, where protease

action removes the sequence after the protein is imported into the ER.

Signal sequences vary in length from 13 to 36 amino acid residues.

All signal sequences have the following features:

1) about 10 to 15 hydrophobic amino acid residues

2) one or more positively charged residues, usually near the amino terminus, preceding

the hydrophobic sequence

3) a short sequence at the carboxyl terminus (near the cleavage site) that is relatively

polar, typically having amino acid residues with short side chains (especially Ala) at

the positions closest to the cleavage site.

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Examples of Signal sequences for protein translocation into ER

Nelson & Cox, Lehninger Principles of Biochemistry, 4th ed., 2005

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Targeting of nuclear proteins


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Targeting of mitochondrial proteins


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Three protein translocators in the mitochondria membrane


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Protein import by mitochondria

The N-terminal signal sequence of the precursor protein is recognized by receptors of the TOM complex.

The protein is thought to be translocated across both mitochondrial membranes at or near special contact

sites. The signal sequence is cleaved off by a signal peptidase in the matrix to form the mature protein.

The free signal sequence is then rapidly degraded (not shown)

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The role of energy in protein import into the mitochondrial matrix


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Translocation of precursor proteins into chloroplast


A) The chloroplast signal sequence initiates

translocation into the stroma through a

membrane contact site by a mechanism similar

to that used for translocation into the

mitochondrial matrix. The signal sequence is

then cleaved off, which initiates translocation

across the thylakoid membrane.

B) Translocation into the thylakoid space or

thylakoid membrane can occur by any one of at

least four routes:

(1) Sec pathway, uses components that are

homologs of Sec proteins, which

mediate protein translocation across the

bacterial plasma membrane.

(2) SRP-like pathway, uses a chloroplast

homolog of the signal recognition particle,

or SRP

(3) ΔpH pathway, it is driven by the H+

gradient across the thylakoid membrane,

and

(4) spontaneous insertion pathway that

seems to require no protein trarnslocatorfor membrane integration.

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The signal hypothesis of protein targeting to ER


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How does a single-pass transmembrane protein become inserted into the ER

In addition to the start-transfer sequence as soluble proteins, however, the protein also contains a stop-

transfer sequence (orange). When the stop-transfer sequence enters the translocator and interacts with a

binding site, the translocator changes its conformation and discharges the protein laterally into the lipid bilayer.


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Integrating of a single-pass membrane protein with an

internal signal sequence into the ER

(A) If there are more positively charged amino acids immediately preceding the hydrophobic core of the start-transfer

sequence than there are following it, the start-transfer sequence is inserted into the translocator in the orientation. The

part of the C-terminal to the start-transfer sequence will therefore be passed across the membrane.

(B) If there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer

sequence than there are preceding it, the start-transfer sequence is inserted into the translocator in the orientation

shown here. The part of the N-terminal to the start-transfer sequence will therefore be passed across the membrane.

Because translocation cannot start before a start-transfer sequence appears outside the ribosome, translocation ofthe N-terminal portion of the protein shown in (B) can occur only after this portion has been fully synthesized.


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Integrating of a double-pass membrane protein with an

internal signal sequence into the ER

An internal ER signal sequence acts as a start-transfer signal and initiates the transfer of

the C-terminal part of the protein. At some point after a stop-transfer sequence has entered the

translocator, the translocator discharges the sequence laterally into the membrane.


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The attachment of a GPI anchor to a protein in the ER

1) Immediately after the completion of protein synthesis, the precursor protein remains anchored in the ER membrane

by a hydrophobic C-terminal sequence of 15–20 amino acids; the rest of the protein is in the ER lumen.

2) Within less than a minute, an enzyme in the ER cuts the protein free from its membrane-bound C terminus and

simultaneously attaches the new C terminus to an amino group on a preassembled GPI intermediate.

3) The signal that specifies this modification is contained within the hydrophobic C-terminal sequence and a few amino

acids adjacent to it on the lumenal side of the ER membrane; if this signal is added to other proteins, they too

become modified in this way.

Because of the covalently linked lipid anchor, the protein remains membrane-bound, with all of its amino acidsexposed initially on the lumenal side of the ER and eventually on the cell exterior.


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“What happen to the newly synthesized in the ER lumen?”

Removing of signal sequences

Folding of polypeptides

Forming of disulfide bond

Glycoprotein formation

Play a key role in protein targeting

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Glycosylation

“Adding of carbohydrate part into protein and there are 2 types of glycosylation”

1. O-linked GalNac 2. N-linked GlcNac

(Ser, Thr)

(Asn)

3030


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A.

B.


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ER

ER & Pre-Golgi Intermediates

Pre-Golgi

Intermediates & Golgi apparatus

Golgi apparatus

Transfer of lipid-liked Glc3Man9GlcNAc2 to Asn in

nascent polypeptides

Removal of all three glucose residues by glucosidase I

& II

Transient reglucosylation by UDP-Glc: glycoprotein

glucosyltransferase

Removal of mannose residues by ER mannosidases

Trimming of glucosylated inermediated by

endomannosidase

Removal of mannose residues by Golgi manosidase Elongation reaction by glycosyltransferase

Adapted from Roth J. Chem. Rev. 2002, 102, 285-303

Process of N-glycosylation

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The role of N-linked glycosylation in ER protein folding

1) The ER-membrane-bound chaperone protein calnexin binds to incompletely folded proteins containing one

terminal glucose on N-linked oligosaccharides, trapping the protein in the ER.

2) Removal of the terminal glucose by a glucosidase releases the protein from calnexin.

3) A glucosyl transferase is the crucial enzyme that determines whether the protein is folded properly or not: if the

protein is still incompletely folded, the enzyme transfers a new glucose from UDP-glucose to the N-linked

oligosaccharide, renewing the protein's affinity for calnexin and retaining it in the ER.

4) The cycle repeats until the protein has folded completely.

Calreticulin functions similarly, except that it is a soluble ER resident protein. Another ER chaperone, ERp57 (notshown), collaborates with calnexin and calreticulin in retaining an incompletely folded protein in the ER.



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1) Misfolded soluble proteins in the ER lumen are translocated back into the cytosol, where they are

deglycosylated, ubiquitylated, and degraded in proteasomes.

2) Misfolded membrane proteins follow a similar pathway.

3) Misfolded proteins are exported through the same type of translocator that mediated their import;

accessory proteins that are associated with the translocator allow it to operate in the export direction.

The export and degradation of misfolded ER proteins



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O-glycosylation

No consensus sequence for Ser and Thr

Consensus for Hyl

Gly – X – Hyl – Y – Arg

Begins with GalNac transferase (N-

acetylgalactosamine)

Mannose common addition to core

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Usually be extracellular proteins or located at cell surface

High structural information content

• molecular recognition

Occurs along the secretory pathway

Often stabilizes structure

Difficult to get crystal structure for more than one or two

carbohydrate residues

Notes for glycosylation

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Utilization of different coats in vesicular traffic

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The postulated role of SNAREs in guiding vesicular

transport

There are at least 20 different SNAREs in an animal cell, each

associated with a particular membrane-enclosed organelles

involved in the biosynthetic-secretory or endocytic pathway.

These transmembrane proteins exist as complementary sets

vesicle membrane SNAREs, called v-SNAREs

target membrane SNAREs, called t-SNAREs,

v-SNARESs and t-SNAREs have characteristic helical

domains.

When a v-SNARES interacts with a t-SNARE, the helical

domains of one wrap around the helical domains of the other

to form stable trans-SNARE complexes, which lock the two

membranes together.

The specificity with which SNAREs interact determines the

specificity of vesicle docking and fusion.

SNAREs specify compartment identity and govern the orderly

transfer of material during vesicle transport.

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Transport from the ER to the Golgi apparatus is mediated by vesicular tubular cluster

1) Vesicular tubular clusters move along microtubules to carry proteins from the ER to

the Golgi apparatus.

2) COPI coats mediate the budding of vesicles that return to the ER from these clusters.

3) The coats quickly disassemble after the vesicles have formed.

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The retrieval pathway to the ER uses sorting signals

(A) The KDEL receptor present in vesicular tubular clusters and the Golgi apparatus, captures the soluble ER resident

proteins and carries them in COPI-coated transport vesicles back to the ER. Upon binding its ligands in this low-pH

environment, the KDEL receptor may change conformation, so as to facilitate its recruitment into budding COPI-

coated vesicles.

(B) The retrieval of ER proteins begins in vesicular tubular clusters and continues from all parts of the Golgi apparatus. In

the neutral-pH environment of the ER, the ER proteins dissociate from the KDEL receptor, which is then returned tothe Golgi for reuse.

4141

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Oligosaccharide processing in the ER and the Golgi

apparatus

Processing begins in the ER with the removal of the glucoses from the oligosaccharide initially transferred to the protein. A mannosidase in the ER membrane

removes a specific mannose. Golgi mannosidase I removes three more mannoses and N-acetylglucosamine transferase I then adds an N-acetylglucosamine,

which enables mannosidase II to remove two additional mannoses. This yields the final core of three mannoses that is present in a complex oligosaccharide.

At this stage, the bond between the two N-acetylglucosamines in the core becomes resistant to attack by a highly specific endoglycosidase (Endo H). Since all

later structures in the pathway are also Endo H-resistant, treatment with this enzyme is widely used to distinguish complex from high-mannose

oligosaccharides. Additional N-acetylglucosamines, galactoses, and sialic acids are added. These final steps in the synthesis of a complex oligosaccharide

occur in the cisternal compartments of the Golgi apparatus. Three types of glycosyl transferase enzymes act sequentially, using sugar substrates that have

been activated by linkage to the indicated nucleotide. The membranes of the Golgi cisternae contain specific carrier proteins that allow each sugar nucleotide to enter in exchange for the nucleotide phosphates that are released after the sugar is attached to the protein on the lumenal face.


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Posttranslational Modification and Targeting of Proteins · 2/1/2017 · Posttranslational Modification and Targeting of ... specific proteins across a membrane from the cytosol into