Posttranslational Modification of Proteins

• This refers to reactions that occur co-translationally (during protein synthesis) or posttranslationally (after protein synthesis)

• There are more than 50 types of posttranslational modifications; we’ll cover a selected group of them

• The most common modification is phosphorylation and dephosphorylation, and we’ll devote future lectures to this topic

• Posttranslational reactions are divided into two main categories– Those that have a signal peptide are targeted to the ER– Those that lack a signal peptide are targeted initially to the cytosol

Protein TargetingNascent polypeptide/ribosome

Cytosol EndoplasmicReticulum

Plasma Membrane

Mitochondria Nucleus

Golgi

SecretoryVesicles

Lysosomes Plasma MembraneCytosolic Pathway Secretory Pathway No signal peptide With a signal peptide

- Signal Seq +

Cytosolic Pathway• Proteins that lack a signal peptide at the amino

terminus are not translocated into the Golgi and are not processed

• These proteins are synthesized on free ribosomes not associated with the rough endoplasmic reticulum

• Final cell location– Cytosol, e.g., hexokinase– Nucleus, e.g., DNA polymerase– Mitochondrion, e.g., cytochrome c

• Other modifications in the cytosol– Acetylation (2C) Prenylation (15 or 20C)– Myristoylation (14 C) Palmitoylation (16C)

NLS (Nuclear Localization Sequence)• The nucleus is surrounded by a nuclear envelope

– Inner nuclear membrane– Outer nuclear membrane– Macromolecules are translocated through a nuclear pore

• All proteins found in the nucleus are synthesized in the cytosol and are translocated through the nuclear pore into the nucleus– Histones, DNA polymerases, RNA polymerases– Transcription factors, splicing factors

NLS (Nuclear Localization Sequence)– Nuclear proteins contain an NLS

• One or two sequences (patches) rich in lysine and arginine

• Can be found anywhere in the protein; at the N-terminus, in the middle, or at the C-terminus

• PKKKRKV is an example; PKNKRKV is inactive

• Attachment of this sequence to normally cytosolic proteins results in the import of such mutated proteins into the nucleus

• The nucleoplasminin story– It is required for chromatin assembly

– It contains two patches that are required for nuclear import

» A Lys-Arg pair

» Four lysines located 10 amino acids further downstream

» KRPAATKKAGQAKKKK, where the key residues are underlined

NLS (Nuclear Localization Sequence)• Mechanism

– Proteins with an NLS bind to importins that take them to the nuclear pore

• The alpha subunit of importin binds the NLS• The beta subunit binds to the nuclear pore

– A Ran GTPase interacts with the protein-importin complex and energizes nuclear translocation with GTP hydrolysis

– The NLS is not removed proteolytically. Why?

• Nuclear Export Sequence (NES)– Newly synthesized ribosomes (RNA and protein) bear nuclear

export sequences– This may involve signals on both proteins and RNA

• Nuclear Retention Signal (NRS)– Found in proteins that bind to immature RNAs in the nucleus– mRNAs that contain introns– Pre-tRNAs

Nuclear Import and Export

• Importin binds to cargo and interacts with nucleoporins

– It is a nuclear import receptor

– It binds to basic NLSs

• RanGTP occurs in the nucleus, RanGDP in the cytosol

• RanGTP dissociates import complexes

• RanGTP forms export complexes

• Ran guanine nucleotide exchange factor (RanGEF) occurs in the nucleus

• RanGTPase activating protein (RanGAP) occurs in the cytosol

• Important points

– Ran is a GTPase

– RanGTP is nuclear

– RanGDP is cytosolic

Mitochondria and Protein Import

• Powerhouse of the cell– Krebs cycle, beta oxidation, pyruvate

dehydrogenase

• Contain DNA, RNA, ribosomes– Synthesize about 20 mitochondrial proteins– Most mitochondrial proteins are synthesized in

the cytosol and imported

Anatomy of the Mitochondionand Protein Import

Import of Proteins • Amino-terminal sequence (10-70 aa)

contains positively charged, ser/thr, and hydrophobic amino acids but no common sequence

• Cyt c has an internal targeting sequence

• Hsp70 keeps proteins in an unfolded state

• Translocation through TOM (transport outer membrane)– The fit is snug during transport

– Ions and other small molecules do not leak across the membrane

• Voltage gradient is required for transport across the inner membrane by TIM

• Presequence is cleaved by a signal protease

• Mit Hsp 70 and 60 aid in translocation and facilitates folding

Insertion of mit membrane proteins• Proteins targeted for mitochondrial membranes

contain hydrophobic stop sequences that halt translocation through the TOM or TIM complexes

Sorting proteins to the intermembrane space• I Through Tom into inner mitochondrial space• II From Tom to Tim with hydrophobic stop

sequences that are cleaved• III Into matrix

– Remove hydrophilic basic sequence– Exposes hydrophobic sequence that directs protein to

inner mitochondrial space

Protein Import and the Peroxisome• Peroxisomes oxidize lipids (fatty acids > 18 carbon atoms)• Peroxisomes contain a single lipid bilayer membrane• Unlike mitochondria, peroxisomes lack DNA• All proteins are encoded by nuclear genes• Peroxisome Targeting Signal (PTS)

– Type I; PTS1• C-terminus• Ser-Lys-Ala (Don’t memorize)

– Type II; PTS2• Rare (4 in humans)• At or near the N-terminus• RLXXXXXH/QL (Don’t memorize)

• Peroxins deliver peroxisomal proteins to the target and insert them into the matrix or the membrane (mechanism ?)

• Take home message: there are peroxisome targeting signals

Membrane Localization Signals I• Posttranslational attachment of lipids to proteins creating

non-membrane spanning integral membrane proteins that will reside on the cytoplasmic surface of the plasma membrane of subcellular membranous organelle

• Myristoylation (14C)– N-terminal processing

• Met-aminopeptidase often removes N-terminal Met• If residue after methionine is a glycine, a myristoyl group can be attached

via an amide linkage, blocking the amino terminal group• The lipophilic myristoyl group can be inserted into the membrane• Several of the alpha subunits of heterotrimeric G-proteins possess this

modification• Not all proteins that are N-myristoylated are attached to membranes

– Myristoyl~CoA + H2N-Gly-protein myristoyl-CO-N(H)-Gly-protein + CoA; the high energy thioester is used to drive the synthesis of the low energy amide linkage

Protein Prenylation II• A 15 carbon farnesyl group or a 20 carbon geranylgeranyl

group is added to proteins that contain a C-terminal CaaX box– C is cysteine– a represents aliphatic residues (not Alanine)– X represents leucine for geranylgeranyl groups and Met, Ser, Ala

for farnesylation

• Ras is farnesylated– Part of the Raf-MEK-ERK pathway– Mutated in 25% of all human cancers– Inhibition of Ras farnesylation is a targeted anticancer target

• The gamma subunit of many G-gamma proteins is geranylgeranylated

• These modifications promote membrane binding• Know what a CaaX box is

Prenylation Sequence of Reactions

Fig. 18-17

Palmitoylation Reactions (16C)

• K-ras, one type of ras, is both farnesylated and palmitoylated

• A protein cysteine is modified as a thioester• Palmitoyl-CoA + protein CysSH protein

CysS~palmitate + CoA

• This concludes the Cytosolic Pathway• Next, the Secretory pathway

Secretory Pathway• Products for secretion, transmembrane proteins, and import into

Golgi/ER/Secretory granules– Preproinsulin (secreted)– Prealbumin (secreted)– Preproinsulin receptor beta subunit (transmembrane)– The pre refers to the signal peptide

• A signal peptide pre sequence at the amino terminus of a protein targets polypeptide/ribosome to the ER– 6-13 hydrophobic proteins near the N-terminus– Usually a positively charged residue nearby– This is the second sequence besides CaaX that you should learn

• Some proteins are cleaved by signal peptidases and are found entirely within the lumen

• Some proteins contain “stop transfer” sequences– These proteins become membrane spanning portions of integral

membrane proteins

Protein Synthesis

• We’ll see this again when we cover amelogenin biosynthesis in enamel formation

• Fig. 18-4

Role of the Signal Sequence and Signal Recognition Particle in Directing a Peptide to the ER (Fig. 18-2)

Fig. 18-2

Insulin Biosynthesis• Synthesized as preproinsulin, the

first such discovered protein• The presequence is cleaved to yield

proinsulin (signal peptidase)• Disulfide bonds form, and the

connecting peptide is cleaved by prohormone convertase

• Carboxypeptidase H finishes the job by cleaving the basic residues

• This yields mature insulin with its A and B chains

• Learn this process; it’s an important prototype

• Fig. 18-3

GPI Anchor Biosynthesis

• GPI: GlycoPhosphatidyl-Inositol anchor

• Fatty acids in membrane• Polar groups in lumen• GPI transamidase

catalyzes the reaction of the amino group with a protein carboxylate to give a new amide bond

• …C(=O)-N(H)-.. + H2N- …C(=O)-N(H)-.. + H2N-

Cotranslational and Posttranslational Modifications

in the ER and Golgi• Most of the modifications produced in

the ER are constitutive (remain until the protein is degraded)

• These modifications take advantage of the unfolded nature of the polypeptide as it enters the lumen of the ER

• Glycosylation Reactions attach carbohydrate: O-linked and N-linked (Fig. 18-7)– O-linked refers refers to the attachment of

sugars to serine or threonine (simple)– N-linked refers to the attachment of sugars

to asparagine (difficult)• High mannose• Complex• Hybrid

Endoplasmic Reticulum and Golgi

• Endoplasmic (inside the cell); reticulum a network• ER, a network inside the cell• Disulfide bond formation occurs in the ER• N-linked oligosaccharide synthesis is initiated in the ER;

trimming and completion occurs in the Golgi• Most O-glycosylation occurs in the Golgi• Attachment of mannose 6-phosphate occurs in the Golgi• Sulfation of secreted proteins occurs in the Golgi• Proline and lysine hydroxylation, alpha amidation, and

vitamin K-dependent carboxylation reactions occur in the Golgi

N-Linked Oligosaccharides (Fig. 18-8)

Activated Carbohydrates

• These serve as carbohydrate donors• As activated sugars, a high-energy bond is used for the

synthesis of a low-energy compound• UDP-Glu, UDP-Gal, UDP-GlcUA, UDP-Xyl, UDP-GlcNAc,

UPD-GalNAc, GDP-Man• CMP-NeuNAc• Dolichol phosphates

• It is not necessary to memorize the following pathways, but you should remember the identity of the activated sugars

Hexosamine Metabolism (Fig. 18-9)

Dolichol phosphate (Fig. 18-10)

Dolichol Phosphate Metabolism (Fig. 18-11)

First Stage of N-Linked Oligosaccharide Synthetsis (Fig. 18-12): Occurs in the ER

Second and Third Stages of N-Linked Oligosaccharide

Synthesis (Fig. 18-13)

• The hydrolysis reactions are unidirectional

• Donation of activated sugars is energetically favorable

• Each reaction is catalyzed by an enzyme that determines the sequence of sugars and the configuration of the glycosidic linkages

• Occurs in Golgi

O-Linked Blood Group BiosynthesisTable 18-2

Blood Group Glycoproteins (Fig. 18-15)

Blood Group Biosynthesis

• Fig. 18-16• Golgi reactions• People with type O blood

groups lack functional A and B genes

• Genes A and B differ by 4 nucleotides which alters the substrate specificity

A: GalNAc transferase

B: Gal transferase

Targeting Enzymes to Lysosomes:A Golgi Process

• Lysosomal proteins contain N-linked oligosaccharides with terminal mannose 6-phosphates

• The addition of phosphate occurs by an unusual mechanism or pathway

• There is a mannose 6-phosphate receptor that recycles between the Golgi and lysosome and participates in the translocation of lysosomal enzymes

Mannose 6-Phosphate Synthesis (Fig. 18-14)

Protein Sulfation Reactions (Fig. 12-12)

Active sulfate: PAPS, phosphoadenosylphosphosulfate

Protein Sulfation

• A Golgi pathway modification

• Fig. 18-6

Procollagen Hydroxylation

• Fig. 18-18• Golgi reactions• Proline and lysine

hydroxylation reactions• Requires vitamin C

– These two hydroxylases– Dopamine beta-hydroxylase– Peptidyl amidating mono-

oxygenase

• The lysyl oxidase Rxn (next slide) inititates collagen cross linking

Lysyl Oxidase Reaction (Fig. 23-12)

Golgi reactions

Protein Amidation

• The amide group is derived from a carboxyterminal glycine

• Ascorbate and oxygen are required

• Golgi reactions• Fig. 18-19

Vitamin K-Dependent Carboxylation Reactions

• Protein-glutamate is carboxylated

• Carbon dioxide, Vitamin K, and oxygen are required

• Several blood clotting factors and other proteins that bind calcium ions contain gamma carboxylation of protein-glutamates

• Golgi reactions

Vitamin K Carboxylation/Oxidation (Fig. 18-20)

Thyroid Hormone Biosynthesis (Fig. 18-21)

Plasma Membrane Topology

• It is not necessary to remember which protein has which type of membrane topology except for GPCRs

Topological Classes• Topological classes I-III have a single pass through the

membrane– I: N-terminus is intraluminal and C-terminus is extraluminal– II: C-terminus is extra, N-terminus is intra; no cleaved sequence– III: Same as I, but no cleavable sequence

• Class IV has multiple passes• Type I, without a signal peptide, contains a 22 aa

hydrophobic stop transfer sequence• Type II and III

– Lack a N-terminal signal peptide– Contain a signal-anchor sequence that functions as an ER signal

sequence and membrane anchor sequence

Type II Membrane

Protein Biosynthesis

• Stop transfer anchor sequence

• C-terminus in the ER lumen or cell exterior

Protein TargetingNascent polypeptide/ribosome

Cytosol EndoplasmicReticulum

Plasma MembraneLipidation with myristate,

palmitate, farnesylate,or geranylgeranylate

MitochondriaBasic aa at N-ter

NucleusBasic amino acids

GolgiGlycosylation, Amidation,

Sulfation, K carbox, Hydroxylation

SecretoryVesicles

LysosomesMannose 6-P

Plasma MembraneStop transfer sequences

- Signal Seq +