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 Targeting
Nascent polypeptide/ribosome
- Signal Seq +
Endoplasmic
Reticulum
Cytosol
Plasma Membrane
Golgi
Mitochondria
Nucleus
Cytosolic Pathway
No signal peptide
Secretory
Vesicles
Lysosomes
Plasma Membrane
Secretory Pathway
With a signal peptide
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)
– Myristoylation (14 C)
Prenylation (15 or 20C)
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 Mitochondion
and 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
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•
•
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)-Glyprotein + 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: GlycoPhosphatidylInositol 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
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•
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•
•
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, UDPGlcNAc, 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 Biosynthesis
Table 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 6phosphates
• 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 monooxygenase
• 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 Targeting
Nascent polypeptide/ribosome
- Signal Seq +
Endoplasmic
Reticulum
Cytosol
Plasma Membrane
Lipidation with myristate,
palmitate, farnesylate,
or geranylgeranylate
Golgi
Glycosylation, Amidation,
Sulfation, K carbox,
Hydroxylation
Mitochondria
Nucleus
Basic aa at N-ter
Basic amino acids
Secretory
Vesicles
Lysosomes
Plasma Membrane
Mannose 6-P
Stop transfer sequences