1. 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

2. Nascent polypeptide/ribosome
Protein Targeting
Nascent polypeptide/ribosome
- Signal Seq +
Endoplasmic
Reticulum
Cytosol
Plasma Membrane
Golgi
Secretory
Vesicles
Mitochondria
Nucleus
Lysosomes
Plasma Membrane
Cytosolic Pathway Secretory Pathway
No signal peptide With a signal peptide

3. 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)

4. 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

5. 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

6. 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

7. 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

8. 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

9. Anatomy of the Mitochondion and Protein Import

10. 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

11. Insertion of mit membrane proteins
Proteins targeted for mitochondrial membranes contain hydrophobic stop sequences that halt translocation through the TOM or TIM complexes

12. 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

13. 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

14. 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

15. 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

16. Prenylation Sequence of Reactions
Fig

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

18. 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

19. Protein Synthesis
We’ll see this again when we cover amelogenin biosynthesis in enamel formation
Fig. 18-4

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

21. 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

22. 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-

23. 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

24. 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

25. N-Linked Oligosaccharides (Fig. 18-8)

26. 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

27. Hexosamine Metabolism (Fig. 18-9)

28. Dolichol phosphate (Fig. 18-10)

29. Dolichol Phosphate Metabolism (Fig. 18-11)

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

31. Second and Third Stages of N-Linked Oligosaccharide Synthesis (Fig
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

32. O-Linked Blood Group Biosynthesis Table 18-2

33. Blood Group Glycoproteins (Fig. 18-15)

34. Blood Group Biosynthesis
Fig
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

35. 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

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

37. Protein Sulfation Reactions (Fig. 12-12)
Active sulfate: PAPS, phosphoadenosylphosphosulfate

38. Protein Sulfation
A Golgi pathway modification
Fig. 18-6

39. Procollagen Hydroxylation
Fig
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

40. Lysyl Oxidase Reaction (Fig. 23-12)
Golgi reactions

41. Protein Amidation
The amide group is derived from a carboxyterminal glycine
Ascorbate and oxygen are required
Golgi reactions
Fig

42. 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

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

44. Thyroid Hormone Biosynthesis (Fig. 18-21)

45. Plasma Membrane Topology
It is not necessary to remember which protein has which type of membrane topology except for GPCRs

46. 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

47. Type II Membrane Protein Biosynthesis
Stop transfer anchor sequence
C-terminus in the ER lumen or cell exterior

48. Protein Targeting Nascent polypeptide/ribosome Endoplasmic Cytosol
- Signal Seq +
Endoplasmic
Reticulum
Cytosol
Plasma Membrane
Lipidation with myristate,
palmitate, farnesylate,
or geranylgeranylate
Golgi
Glycosylation, Amidation,
Sulfation, K carbox,
Hydroxylation
Mitochondria
Basic aa at N-ter
Nucleus
Basic amino acids
Secretory
Vesicles
Lysosomes
Mannose 6-P
Plasma Membrane
Stop transfer sequences