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

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

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

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

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 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 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