Principles of Molecular and Cellular Biology Unit 3

Three phases of translation

1. Initiation

2. Elongation

3. Termination

Translation of mRNA requires

3 types of RNAs (mRNA, tRNA, and rRNA), energy (ATP, GTP), and proteins

Eukaryotic mRNA

5' UTR > 8 nucleotides

Coding region/open reading frame (defined by the first "strong" AUG

3' UTR

"Strong" AUG

In most mRNAs, the first AUG from the cap is "strong," and it serves as the start codon

10% mRNAs: first "strong" AUG is not the first AUG

tRNA characteristics

1. tRNAs are synthesized and modified in the nucleus

2. tRNA modifications are: base modifications, shortening of the 5' end, and adding CCA to 3' end

3. tRNAs are exported to the cytoplasm where they are aminoacylated

4. Loops of tRNAs are important for interactions with proteins and the translated mRNA

Amino acid-tRNA specificity

Attachment of the amino acids to their respective tRNAs is highly specific

Each amino acid has its own aa-tRNA synthetase that recognizes the aa

Proofreading activity of aminoacyl-tRNA synthetases

Ensures correct charging of tRNAs (cleaves bond between the aa and the incorrectly charged tRNA)

3rd position wobble of codon

FIrst and second bases of the codon form standard base pairs (GC and AU), but the third base of the codon can form non-standard pairs with the anticodon (wobbling)

Assembly/maturation of rRNA

The 28S rRNA has catalytic activity critical for peptide bond formation during translation

Three rRNAs (except the 5S rRNA) are cut from sing pre-rRNA, the nucleolus is a ribosome factory where rRNAs are produced, where ribosome assembly starts

Translation initiation

1. Formation of the 43S preinitiation complex

2. mRNA activation

3. Assembly of the 80S ribosome

Result of translation initiation is a correctly positioned 80S complex with its Met-tRNA at the P site (complex is ready for elongation)

Translation polypeptide chain elongation

1. eEF1alpha binds GTP and an aa-tRNA (ternary complex formed)

2. Codon at the A site recruits the complementary aa-tRNA-eEFIalpha-GTP complex

3. Correct codon-anticodon pairing induces hydrolysis of GTP by eEF1alpha (leads to release of eELF1alpha by conformational change)

4. In new conformation state of ribosome, the two aa-tRNAs are close together and the large rRNA catalyzes formation of peptide bond

5. The 80S complex binds eEF2-GTP, the GTP is hydrolyzed by EF2, and this provides energy for translocation (tRNAi ends up in the E site and dipeptidyl-tRNA is at the P site, and the ribosome is ready to bind the next ternary complex at the A site)

Termination of translation

1. eRF1 binds to eRF3-GTP (yet another ternary complex is formed)

2. eRF1-eRF3-GTP recognizes the stop codon

3. GTP is hydrolyzed by eRF3

4. GTP hydrolysis by eRF3 causes conformation changes and this leads to the cleavage of the peptidyl-tRNA bond and the release of the polypeptide

5. A few eIFs and the ATPase ABCE1 are required for the dissociation of ribosomal subunits from the mRNA, for the dissociation of the tRNA from the P site, and for the release of eRF1 and eRF3

The poly(ribo)some

Most mRNA molecules are translated several times, many ribosomes can translate a single mRNA molecules nearly simultaneously

These mRNA and many-ribosome structures are called the poly(ribo)somes

Cellular stresses that lead to regulation of translation

ER stress (incorrectly folded proteins accumulate in the ER), not enough amino acids and other metabolic stresses, viral infections, and apoptosis

Global regulation of mRNA translation

1. Phosphorylation of eIF2

2. Phosphorylation of eIF4E Binding Protein (4E-BP)

3. Proteolytic cleavage of eIF4G and PABPC by caspase-3 or viral proteases (during apoptosis and viral infection)

mRNA-specific regulation of mRNA translation

Regulation through interactions between proteins and unique structures in mRNAs

1. Upstream open reading frame (uORF)

2. Internal Ribosome Entry Site (IRES)

eIF2 phopshorylation by eIF2 kinases

Leads to global inhibition of translation: Phosphorylation of the α subunit of the heterotrimeric protein eIF2 at a Ser residue (Ser51) blocks the GDP to GTP exchange reaction by eIF2B, which is the GEF of eIF2; result is concentration of the ternary complex of translation initiation is reduced

eIF2 kinases and cellular stresses

4 eIFK2 kinases are activated by different stresses

1. Heme depletion: EIF2AK1 (heme-regulated inhibitor, HRI kinase)

2. dsRNA: EIF2AK2 (dsRNA activated protein kinase, PKR)

3. ER stress: EIF2AK3 (pancreatic eIF2alpha kinase, PERK)

4. Amino acid depletion: EIF2AK4 (general contron non-derepressible 2 GCN2)

PERK mechanism

PERK is activated when unfolded proteins sequester the chaperone BiP in the ER lumen

ER stress > BiP is sequestered by misfolded proteins in ER lumen > PERK releases BiP, leading to PERK dimerization and activation of its kinase, PERK phosphorylates itself and eIF2 > phosphorylation causes global inhibition of translation but translation of ATF4 increases > ATF4 induces expression of many other stress response proteins

GCN2 mechanism

GCN2 is activated by non-acylated tRNAs, concentration non-acylated tRNAs increases in the cytosol during amino acid starvation

GADD34

GADD34 provides a scaffold for interaction of protein phosphatase 1 (PP1) and P-eIF2alpha

ER stress > eIF2alpha kinase > eIF2alpha is phosphorylated to P-eIF2alpha by PP1, leads to ATF4 translation increase which upregulates GADD34 transcription

ATF4 mRNA translation when eIF2 is phosphorylated

The ATF4 contains 2 uORFs besides the ATF4-coding sequence

The first uORF is very short whilethe second uORF overlaps with the ORF of ATF4, the second uORf and the ORF of ATF4 are not in frame

Model for uORF-dependent regulation of ATF4 translation

1. 40S remains on the mRNA at the stop of uORF1, and it continues to move along the mRNA

2. 40S becomes competent for translation reinitiation faster if the concentration of the ternary complex (TC) is high in the cytosol

No stress: high TC concentration

Stress: low TC concentration

The 40S can remain on the mRNA at the stop because the uORF1 of ATF4 is very short (too long: 40S falls off)

Stimulation of translation through 4E-BP phosphorylation

Rapamycin blocks metazoan target of rapamycin (mTOR)

The non-phosphorylated eIF4E-binding protein (4E-BP) inhibits the interaction between eIF4E and eIF4G, thus translation is repressed

When nutrients are available: mTOR phosphorylates 4E-BP and 4E-BP releases eIF4E (promotes translation initiation)

Amino acid deprivation

Amino acid depreivation regulates the availability of ternary complex and eIF4G for translation initiation, amino acid deprivation regulates both GCN2 and mTOR

IRES (internal ribosome entry site)

IRES is an mRNA sequense with a strong 3D structure that recruits the 43S complex directly without using the 5' cap or eIF4E

IRES can recruit the 43S complex to the "middle" of the mRNA, IRES escapes regulation by 4E-BP and mTOR

IRES and picornaviruses

IRES was discovered in picornaviruses: the picornavirus RNA does not have a cap, picornaviruses don't need eIF4E for replication, and picornavirus replication is not inhibited by rapamycin

IRESes and eIFs

Picornavirus IRES:

1. Viral proteinase cuts eIF4G in such way that it can no longer bind to eIF4E

2. The central piece of eIF4G is recruited to the IRES (this is followed by the recruitment of eIF4A and eIF4B)

3. 43S assembles with the 4G central piece at the IRES

4G: central piece of eIF4G after cleavage

Endoplasmic Reticulum

ER is a membrane delimited organelle composed of the nuclear envelope and a network, functionally and visually, the ER can be divided into rough and smooth ER

Rough vs smooth ER

Rough: secretory proteins are synthesized at the rough ER

Smooth ER: synthesis of fatty acids, phospholipids, steroid hormones, detoxification, Ca2+ storage

Protein targeting

Protein targeting to the right organelle requires targeting sequences, which are recognized by organelle-specific receptors

ER is the only entry point of the secretory pathway for newly synthesized proteins

Mitochondrial localization signal

~30 aa peptide at the N-terminus forms an amphipathic alpha-helix (positively charged aa on one side, hydrophobic on the other)

Nuclear localization signal

Short sequences of positively charged aa (K and R)

Two experiments demonstrating secretory proteins entering ER co-translationally

1. Cell-free protein synthesis, microsomes present (rough microsomes generated through mechanical homogenization of cells that contain a lot of rough ER)

2. Cell-free protein synthesis, no microsomes present (ribosomes stripped from the ER and used for synthesis)

Signal-recognition particle (SRP)

Secretory proteins are targeted to the ER by a signal sequence or a transmembrane domain, these ER targeting regions are recognized by cytosolic receptor (SRP)

SRP is a cytosolic multi-subunit ribonucleoprotein, a hydrophobic groove in one of the SPR subunits binds to the ER signal sequence, it also binds to the ribosome

ER signal sequence

16-30 aa region with many hydrophobic side chains, ER signal sequences are at the N-terminus, they are less hydrophobic than transmembrane regions, and are cleaved

The first transmembrane domain (most N-terminal) of a protein can also act as an ER-targeting sequence (but it is not cleaved)

Protein translocation into the ER

1. SRP slows down protein translation (ribosome has time to dock to the ER and is mediated by interaction of SRP and its ER resident receptor)

2. Two GTPase active sites are reconstituted at the interface and two GTP molecules are hydrolyzed, the ribosome is released from the SRP and placed onto the translocon

3. The signal peptide is recognized and cleaved by the signal peptidase

Translocon structure

Translocon consists of alpha, beta, gamma subunits, it can open longitudinally between TM5 and TM6

Longitudinal gate: oscillates between open and closed conformations (can move laterally into lipid environment if polypeptide chain is hydrophobic)

The plug: has positive charges on top, needs to move sideways to allow protein transport

The channel: can accomodate a maximum two linear segments of a polypeptide chain (one in the lumen and one in the hydrophobic opening of the longitudinal gate)

Glycosylphosphatidylinositol (GPI)-anchored proteins

GPI serves as a membrane anchor for many eukaryotic cell surface proteins that are involved in diverse biological functions such as immune recognition, signal transduction, and cell adhesion

Prevention of aggregation of nascent proteins in the ER

Prevention of nascent proteins is prevented in the ER by:

1. Covalent modifications: cleavage, glycosylation, formation of disulfide bonds

2. Non-covalent modifications: folding (often with the help of chaperons that prevent protein aggregation by "hiding" the hydrophobic regions of polypeptides)

Mechanisms to promote correct protein folding

1. Prevent protein aggregation: hydrophobic pockets of chaperons hide the exposed hydrophobic regions of nascent proteins, and glycosylation can make hydrophobic regions hydrophilic

2. Slow down folding: chaperons slow down folding

3. If a protein is badly folded: denature and re-nature it

Classes of ER chaperons

1. Some heat shock proteins including BiP

2. Calnexin and calreticulin (exclusive to the ER)

3. Protein disulfide isomerases (PDI are exclusive to the ER): they assist in the formation and rearrangement of disulfide bonds

4. Peptidyl-prolyl isomerases (PPI): enzymes that facilitate rotation arounf peptidyl-prolyl bonds

BiP

BiP binds to exposed hydrophobic regions of nascent proteins and prevents protein aggregation

BiP uses the energy from ATP to induce local unfolding in the substrate, this helps proteins to overcome energy barriers in the process of finding their native conformation

Calnexin and calreticulin

Calnexin and calreticulin are related but non-redundant ER chaperones, their lectin sites recognize oligosaccharide groups on folding glycoproteins

Calnexin and calreticulin recruit PDIs to reduce the energy barrier for refolding (they do not use ATP)

Glycosylation in the ER

Glycosyl chains are indicators of the folding status of the protein in the ER and glycosylation promotes interactions with the ER chaperons CLR and CNX

N-linked glycosylation

Through the amide group of Asn-X-Ser/Thr motif

1. Very common (most secreted proteins)

2. Complex, branching oligosaccharides

3. Not all Asn-X-Ser/Thr are glycosylated (quick folding can prevent glycosylation)

O-linked glycosylation

Through the -OH if Ser, Thr (no special recognition motif)

Addition of preformed oligosaccharides to proteins

N-linked glycosylation in the ER

1. Assembly of an oligosaccharide precursor on a lipid (called dolichol)

2. Transfer to a Asn-X-Ser/Thr motif in a secretory protein

3. Modifications of the oligosaccharide chain in the ER

4. Further modifications of the oligosaccharide chain in the Golgi (only the 5 core residues do not change in the ER and Golgi)

N-linked glycosylation-dependent interactions with CLR/CNX and UGGT

Upon release from CLR/CNX, the glycoprotein is deglucosylated by glucosidase II, if folding is complete, the glycoprotein is not a substrate for UGGT (UGGT is a folding sensor)

If folding is incomplete, the glycoprotein becomes a substrate for UGGT, UGGT reattaches a single glucose to the M9 chain of the non-native conformer; thus, the glycoprotein can interact with CLR and CNX again

Mannosidase I-dependent exit from the CNX/CLR-GII-UGGT cycle

Time to time, mannosidase I (ERManI) removes glycoproteins from the CLR/CNX-GII-UGGT cycle even if they are not fully folded

UGGT does not recognize M8 glycoproteins, UGGT has higher activity toward misfolded glycoproteins than ERManI, but misfolded proteins are "sooner or later" removed from the CLR/CNX-GII-UGGT cycle by ERManI-catalyzed cleavage of mannose (this is the "mannosidase clock")

ERAD: ER-associated degradation of incorrectly folded secretory proteins

Proteins are recognized for reverse translocation to the cytosol based on long retention time in the ER and incomplete folding

Misfolded proteins are retro-translocated into the cytosol through a channel complex, misfolded proteins are ubiquitinated and degraded in the cytosol

Polyubiquitination and proteasome-dependent degradation

1. ATP-dependent activation of ubiquitin

2-3. Ubiquitin conjugation to an enzyme, and ubiquitin ligation to a Lys (K) in the cytosolic target protein

4. Polyubiquitination

5. Recognition of (Ub)p by the proteasome and ATP-dependent degradation

ER stress/unfolded protein response (UPR) pathways: PERK pathway

ER stress > BiP is sequestered by misfolded proteins > PERK releases BiP, this leads to PERK dimerization and activation of its kinase domain, PERK phoshporylates itself and eIF2 > phosphorylation of eIF2 causes gloval inhibition of translation but the translation of ATF4 mRNA increases > ATF4 is a transcription factor, ATF4 induces expression of many stress response genes

ER stress/UPR pathway: the IRE1-XBP1 pathway

BiP is sequestered by misfolded proteins in the ER lumen > IRE1 releases BiP, IRE1 dimerizes > this causes auto- and cross-phosphorylation in the dimer, leading to activation of IRE1 endonuclease activity > IRE1 splices out a 26-nucleotide region from the XBP1 mRNA > XBP1s activates the transcription of genes that contain ER stress elements (ERSE) in their promoter

ER stress/UPR pathways: ATF6 pathway

ER stress > BiP is sequestered by misfolded proteins > ATF6 releases BiP, and this unmasks Golgi localization signals in ATF6 > ATF6 is transported to the Golgi where it is cleaved on the cytosolic side by a Golgi resident enzyme > this releases a transcription factor (cytosolic domain of ATF6), which translocates into the nucleus and binds to several different promoter elements in ER stress response genes

Functions of biological membranes

1. Limit cells and organelles (they maintain unique ionic environments, separate unique sets of proteins from the rest of the cell)

2. Anchor and organize proteins

3. Provide substrate for signal transduction events

Lipid components of membranes

Glycerophospholipid, sphingolipids (phosphosphingolipids, glycosphingolipids), cholesterol

Cholesterol in bilayer

Cholesterol is inserted into bilayer membranes

with its hydroxyl group oriented toward the

aqueous phase & its hydrophobic ring system

adjacent to fatty acid chains of phospholipids

Biological membranes as 2-D fluids at 37C

Composition:

1. cholesterol content increases membrane

rigidness

2. as the amount of saturated fatty acids

increases, rigidness also increases because the

membrane is "better" structured

3. Shorter FA tails: more fluidity

4. More sphingolipids cause increased

rigidness because they form H-bonds with

cholesterol

Liposomes vs. micelles

Natural phospholipids form liposomes, not micelles

Steep turns in the membrane are made

possible by the small size of the PE

head group

Solubilization of membranes and TM proteins with detergents

Detergents can be ionic (they denature and

solubilizes proteins), or non-ionic (functionality of

some TM proteins can be preserved)

Lipid bilayer symmetry

The lipid bilayer is symmetrical only in the ER, membranes 'distal' to the ER are asymmetric

The 'faces' of cellular membranes are preserved during vesicular transport

EC leaflet: PC, sphingolipids, and cholesterol

IC leaflet: PS, PEA, PI, and cholesterol

Synthesis of glycerophospholipids

Fatty acids (FA) are synthesized in the cytosol, ER flippases can move glycerophospholipids until equilibrium is reached between the two leaflets of the ER membrane, they do not use ATP

In the Golgi and plasma membrane, flippases and floppases

generate asymmetric glycerophospholipid distribution

Sphingolipid synthesis

Sphingolipids are produced and distributed asymmetrically between the membrane leaflets in the Golgi

Mechanism of phospholipid transport from ER to other organelles

1. Vesicular transport (secretory pathway)

2. Non-vesicular transport: mainly at ER contact sites by Lipid Transport Proteins (LTPs)

Protein composition of membrane bilayer

1. Integral membrane proteins: transmembrane proteins and monotopic membrane proteins

2. Lipid anchored membrane proteins: C-terminal GPI (glycosyl-phosphatidyl inositol) anchor

3. Peripheral membrane proteins are attached to the EC or IC surface (they are water-soluble proteins)

Methods to study lateral movement of proteins and lipids in the plasma membrane

1. Fluorescence Recovery After Photobleaching (FRAP): fluorescent labeling of membrane proteins/lipids, photobleaching of an area with laser scans, measure fluorescence recovery in the scanned areas

2. Exoplasmic single particle tracking: requires fluorophore labeling of a membrane-embedded molecule and video recording at high frame rate

"Picket fence" model

Explains the restricted movements of phospholipids between membrane "compartments", trans-membrane proteins are the pickets, increased packing of membrane lipids near transmembrane proteins leave little space for phospholipids to cross from one compartment to the next

Lipid rafts

Lipid rafts are detected based on their:

1. Insolubility in non-ionic detergents at 4C

2. Special lipid composition: sphingolipid and cholesterol rich, GPI-anchored protein rich, gel-like, and thick

3. Low buoyant density on sucrose gradients

Lipid rafts are highly dynamic domains that function as signaling and sorting platforms

Secretory pathway

ER>Golgi>secretory vesicles/granules

Pulse-chase experiment in pancreatic acinar cells

Pancreatic exocrine cells: distribution of autoradiographic grains at the end of a 3 minute pulse, 86% of grains are associated with the ER

At the end of 37 minutes: distribution of autoradiographic grains following three minute pulse shows condensing vacuoles

VSV-G protein trafficking study

Non-radioactive pulse-chase assay used to study VSV-G (stomatitis virus G, transmembrane protein that folds in a temp. sensitive manner) protein trafficking along the secretory pathway

VSV-G*-GFP transfection and fluorescence microscopy shows 40C is non-permissive temp (misfolded, cannot exit ER) while 32C is permissive temp (correctly folded, can exit from ER)

Protein glycosylation status

Glycosylation status is informative about the ER versus "post-ER" localization of secretory proteins

Secretory protein glycosylation occurs in the ER and is completed in the Golgi along the eukaryotic secretory pathway

Microbial glycosidases and secretory protein localization

Microbial glycosidases can reveal the ER vs. "post-ER" localization of secretory proteins

1. N-glycosidase F removes all N-linked oligosaccharide chains

2. Endo-glycosidase H remove oligosaccharide chains that are added in the ER and not yet modified in the medial or trans Golgi

Summary: shift with endo-H treatmen=ER localization while no shift with endo-H but shift with N-glycosidase-F treatment=Golgi or post-Golgi localization

Genetic approach to analyze mechanism of protein trafficking

1. Random mutagenesis (creates mutants defective of protein secretion, "sec" mutants)

2. Incubate @ non-permissive temp

3. If secretory proteins remain in the cell, the cell becomes denser (faster sedimentation)

4. Sec mutants accumulate the normally exocytosed proteins at non-permissive temp, this alters the morphology of secretory organelles

Protein trafficking biochemical approach: GTPyS

GTPyS (inhibitor of GTPase, binds to GTPase making it unable to hydrolyze, always active) leads to accumulation of coated vesicles

Protein trafficking biochemical approach: NEM

NEM (inhibitor of NSF) leads to accumulation of non-coated vesicles

General model of vesicular transport processes

1. Activation of a GTPase on a donor membrane by a GEF

2. Recruitment of coat proteins, GTPase-activating protein

3. Coat polymerization deforms the membrane

4. Scission (dividing/cutting)

5. Hydrolysis of GTP by the vesicle-attached GTPase leads to uncoating, vesicle becomes attached to the acceptor membrane via tethers

6. Vesicle fuses through a v-SNARE-T-snare mediated mechanism

7. NSF (NEM-sensitive factor) unwinds the SNARE complex

Identification of NEM sensitive factor (NSF) and NSF-interacting proteins

(ion-exchange activity) NSF is a chaperone-like protein with ATPase activity

alpha-SNAP (soluble NSF association protein) is an adapter protein required for NSF-SNARE interaction

SNAREs: SNAP receptors

v-SNAREs (vesicle) can bind to their cognate t-SNAREs (target membrane)

Specific v-SNARE-t-SNARE recognition contributes to the correct targeting of transport vesicles, and it is essential for the fusion of vesicles and their target organelle

NEM effect

In the absence of NSF activity, multiple rounds of fusion of the vesicle pool can occur, with the consequent accumulation of cis-SNARE complexes

This disrupts further vesicle fusion because "free" SNAREs are depleted

ER exit sites

Ribosome-free areas of rough ER where most ER-derived vesicles bud

There are 50-70 ERES per cell and ERES are relatively stable (>10 min life-time)

Formation of COPII vesicles at ER exit sites

1. Sar1-GDP binds to its ER-specific GEF (Sec12), Sec 12 catalyzes the release of GDP from Sar1, allowing a GTP molecule to bind to Sar1

2. GTP binding alters the conformation of Sar1: its amphipathic N-terminal α-helix becomes exposed.

This N-terminus anchors Sar1-GTP to the cytosolic leaflet of the ER membrane

3. Sar1-GTP recruits the first layer of the COPII coat

4. Sec23 recruits a v-SNARE

5. Sec24 recruits transmembrane cargo proteins and transmembrane cargo receptors

6. The Sar1-GTP-Sec24-Sec23 pre-budding complex recruits the second layer of the COPII coat, the assembled coat pinches off the vesicle

Cargo selection for COPII vesicles

ER export signals of transmembrane cargo proteins are located in their cytoplasmic domains, soluble cargoes are attached to transmembrane cargo receptors which have their own ER export signals

COPII vesicles targetting to ERGIC

ERGIC is close to ER exit sites, and ERGIC is a site of antero- and retrograde sorting

COPII vesicle tethering to ERGIC and fusion

(Occurs after formation of COPII vesicles at ER exit sites)

7. Sar1 hydrolyzes GTP, and this destabilizes the coat

8. The vesicle sheds its coat

9. The vesicle becomes tethered to the

ERGIC / cis-Golgi by a tether and Rab1-

GTP

10. Interaction of v-SNARE and t-SNAREs

causes membrane fusion

11. α-SNAP-NSF unwinds the v/t-SNARE

complex

COPII defects

COPII defects lead to swelling of ER and disintegration of Golgi