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UD / Biology / BIOL 401 / What are the hormones that bind to nuclear receptors in the cytosol or

What are the hormones that bind to nuclear receptors in the cytosol or

What are the hormones that bind to nuclear receptors in the cytosol or

Description

School: University of Delaware
Department: Biology
Course: Molecular Biology of the Cell
Professor: Salil lachke
Term: Fall 2016
Tags: molecularbiology, Biology, splicing, and membranetransport
Cost: 50
Name: Exam_3.pdf
Description: This study guide covers everything for our next exam (very detailed).
Uploaded: 11/12/2016
32 Pages 64 Views 3 Unlocks
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Splicing


What are the hormones that bind to nuclear receptors in the cytosol or nuclear?



Lipid-soluble  

- Lipid-soluble hormones are extracellular signals that regulate the activities of  transcription factors

o They can diffuse through the plasma and nuclear membranes and interact  directly with the transcription factors they control

o This is due to their hydrophobicity 

Nuclear receptors  

- Activators of transcription with three domains

- Example of hormones that bind to nuclear receptors in the cytosol or nuclear o Cortisol  

o Retinoic acid

o Thyroxine  

- Each nuclear receptor has three functional region  


What are the three functional regions of the nuclear receptor?



o 1. Variable region

▪ N-terminal region  Don't forget about the age old question of What is the meaning of tolerance in substance use disorder?

▪ Activation domain  

▪ May contain one or more activation domain

o 2. DNA-binding domain

▪ Contains a repeat of the C4 Zinc finger motif  

o 3. Ligand-binding domain  

▪ C-terminal end

▪ Hormone-binding domain  

- Hormones binding to a nuclear receptor regulates its activity as a transcription factor - Fusion proteins demonstrate hormone-binding domain of GR (Glucocorticoid receptor)  proteins mediates translocation to the nucleus in the presence of hormone - Without hormone, GR cannot be transported into the nucleus  


What is the meaning of the ligand-binding domain?



If you want to learn more check out What is the meaning of hot jupiters in astronomy?

- In cells that expressed β-galactosidase alone 

o The enzyme was localized to the cytoplasm in the presence and absence of the  glucocorticoid hormone

- Cells that expressed fusion protein (β-galactosidase and entire glucocorticoid receptor) o Without the hormone, the fusion protein was present in the cytoplasm o With the hormone, the fusion protein was transported to the nucleus

- Cells that expressed fusion protein (β-galactosidase and only the glucocorticoid receptor  ligand binding domain)

o Without the hormone, the fusion protein was present in the cytoplasm o With the hormone, the fusion protein was transported to the nucleus

Regulation by transcription initiation  

- This is one of many examples where transcription initiation is controlled - The glucocorticoid receptor associates with two inhibitors  We also discuss several other topics like What is significant about the ediacaran biota?

- The inhibitors keep the GR in a folded state  

- When the hormone is present, it diffuses through the plasma membrane and binds to  the ligand-binding domain, it causing a conformational change that allows it to  translates to the nucleus  

o Activates transcription by interacting with chromatin-remodeling and histone  acetylase complex

Steps in processing of pre-mRNA

- In long pre-mRNAs, splicing occurs concurrently with transcription, before cleavage at 3’ end

- mRNA-template DNA hybrids help visualize spliced mRNAs

Alternative cleavage of pre-mRNA

- Two types of synthesis of IgM  

o Secreted IgM

o Membrane IgM

- The specific mRNA that is made, depends on which Poly (A) site is selected during  processing of primary transcript

- Secreted IgM 

o Polyadenylation at upstream site  

o If the upstream poly(A) is used, the resulting mRNA includes the entire Cu4 exon  and specifies the secreted form of the µ chain  

- Membrane IgM 

o Polyadenylation at downstream site  Don't forget about the age old question of How does winning the lottery tend to effect people's happiness?

o If the downstream poly(A) is used, the splicing to the transmembrane exons,  yielding a mRNA that encodes the membrane-bound form of the µ chain  - Cu4

o Codes for 4th µ constant region

- µs

o Codes for secreted IgM

- TM1 and TM2

o Code for transmembrane domains

Alternative Splicing  

- Permits different proteins to be made in different tissues

- Alternative RNA splicing increases the number of protein isoforms expressed from a  single eukaryotic gene.

- Fibronectin is a multifunctional extracellular matrix protein that is secreted from  fibroblasts (connective tissue cells) and hepatocytes (liver cells).  

- The ∼75-kb fibronectin gene contains multiple exons (and introns) that are differentially  spliced in different cell types.  

- Fibroblasts produce fibronectin mRNAs that contain exon EIIIA and EIIIB - Alternative splicing of the fibronectin primary transcript in the hepatocytes, the major  type of cells in the liver, yields mRNAs that lack the EIIIA and EIIIB exons If you want to learn more check out Why did charlotte perkins gilman wrote the yellow wallpaper?

- Hepatocytes splice the EIIIA and EIIIB exons and their flanking introns out of the  fibronectin mRNA. Hepatocyte fibronectin cannot bind to fibroblasts and instead  circulates in the bloodstream and participates in formation of blood clots through fibrin binding domains

- Nearly 90% of all human’s genes are expressed as alternatively spliced mRNAs  Self-splicing introns

- Group 1  

o Protozoans

o Other single cell organisms

o Use guanosine as a cofactor and can fold by internal base pairing to juxtapose  the two exons that must be joined  

o G – not part of the RNA chain

- Group II 

o Mitochondria,

o Chloroplasts

o Use A instead of G

o A – part of the RNA chain  

- Both Group 1 and II

o RNA function as a ribozyme, an RNA sequence with catalytic ability  

o No protein required  

- Spliceosome  

o Protein required  Don't forget about the age old question of What is the part-whole fallacy / fallacy of composition

- Both Group 1, Group II and spliceosome  

o Involve two transesterification reactions, which require no input of energy Properties and chemistry of splicing reaction

- The branch-point (A) is usually 20-50 bases from the 3’ splice site

- During the formation of a mature, functional mRNA, the introns are removed and the  exons are spliced together

- For short transcription units, RNA splicing often follows cleavage and polyadenylation of  the 3’end of the primary transcript  

- For long transcript units containing multiple exons, splicing of exons in the nascent RNA  begins before transcription of the gene is complete

- First Transesterification reactions  

o The ester bond between the 5’ phosphorus of the intron and the 3’ oxygen of  exon 1 is exchanged for an ester bond with the 2’ oxygen of the branch-point A residue  

- Second Transesterification reactions 

o The ester bond between the 5’ phosphorus of exon 2 and exon 3’ oxygen of the  intron is exchanged for an ester bond with the 3’ oxygen of exon 1, releasing the  intron as a lariat structure and joining the two exons  

Splicing requires the assembly of a spliceosome

- 5 Splicing snRNPs + Other proteins assemble on pre-mRNA

- After U1 base pairs with the consensus 5’ splice site, SF1 binds the branch-point A; U2AF associates with the polyprimidine tract and 3’ splice site; and the U2 snRNP associates  with the branch-point A via base-pairing interactions  

- A trimeric snRNP complex of U4, U5, and U6 joins the initial complex to form the  spliceosome

- Rearrangements of base-pairing between snRNAs converts Spliceosome into  catalytically active conformation and destabilizes U1, U4, which are released, this leads  to the 1st catalytic reaction

- The catalytic core, thought to be formed by U6 and U2, then catalyzes the first  transesterification reaction, forming the intermediate containing a 2’, 5’ - phosphodiester bond

- Following further rearrangements between the snRNPs, the second transesterification reaction joins the two exons by a standard 3’, 5’ -phosphodiester bond and releases the  intron as a lariat structure as well as the remaining snRNs

- The excised lariat intron is converted into a linear RNA by a debranching enzyme  SnRNA in snRNPs base pair with pre-mRNA during splicing  

- Five U-rich snRNAs (U1, U2, U4, U5, U6) participate in pre-mRNA splicing  o Ranging in length from 107-210 nucleotides  

o 6-10 proteins associated with each snRNA

- In vivo experiments showed that base pairing-disrupting mutations in the pre-mRNA 5’  splice site also black RNA splicing  

o Splicing can be restored by expression of a U1 snRNA with a compensating  mutation that restores base pairing to the mutant pre-mRNA 5’ splice site

Splicing and Nuclear Transport

Spliceosome forms from two adjacent cross-exonal complexes  

- Average length of an exon is the human genome is about 150bases

- Average length of an intron is about 3500 bases

- A family of RNA-binding proteins, the SR proteins, interact with sequences within exons  called exonic splicing enhancers 

- SR proteins contain several protein-protein interaction domains rich in arginine (R) and  serine (S) residues, called RS domains

- When bound to exonic splicing enhancers, SR proteins mediate the cooperative binding  of U1 snRNP to a true 5’ splice site and U2 snRNP to a branch point through a network  of protein-protein interactions that span an exon

- The complex of SR proteins, snRNPs, and other splicing factors that assemble across an  exon, which has been called a cross-exon recognition complex, permits precise  specification of exons in long pre-mRNAs

- The correct 5’ GU and 3’ AG splice sites are recognized by splicing factors on the basis of  their proximity to exons  

- The presence or absence of specific splicing factors can regulate the splicing process  (defects in which result in disease – spinal muscular atrophy).

Alternative splicing controls sex determination in Drosophila

Sxl protein 

- The sex-lethal (Sxl) protein, encoded by the sex-lethal gene is present only in female  embryos  

- Early in development, the sxl gene is transcribed from a promoter that functions only in  female embryos  

- Later in development, this female-specific promoter is shut off, and another promoter  for sex lethal becomes active in both male and female embryos  

- Male embryos  

o In the absence of early sxl protein, exon 2 of the sxl pre-mRNA is spliced to exon  3 to produce an mRNA that contains a stop codon early in the sequence  

o Males produce no functional sxl protein either early or later in development  - Female embryos

o Functional sxl mRNA is produce

o Sxl binds to a sequence in the pre-mRNA near the 3’ end of the intron between  exon 2 and exon 3, thereby blocking the proper association of U2AF and U2 snRNP with the adjacent 3’ splice site used in males

o As a consequence, the U1 snRNP bound to the 5’ end of the intron between exon  2 and exon 3 assembles into a spliceosome with U2 snRNP bound to the branch

point at the 3’ end of the intron between exon 3 and 4, leasing to the splicing of  exon 2 to exon 4, and the skipping of exon 3

o The branch site for sxl is called intronic silencer 

Tra protein 

- In male embryos  

o No sxl is expressed, exon 1 is spliced to exon 2, which contains a stop codon that  prevents synthesis of a functional Transformer (Tra) protein

- In female embryos  

o Binding of sxl protein to an intronic splicing silencer at the 3’ end of the intron  between exons 1 and 2 blocks binding of U2AF at this site  

o When Sxl is bound, U2AF binds to a lower-affinity site farther 3’ in the pre mRNA; as a result, exon 1 is spliced to this alternative 3’ splice site, causing  skipping of exon 2 with its stop codon

o The resulting female specific transformer mRNA, which contains additional  constitutively spliced exons, is translated into functional Tra protein

Dsx gene 

- In female embryos, a complex of Tra and two constitutively expressed SR proteins, Rbpl  and Tra2, directs the splicing of exon 3 to exon 4 and also promotes  

cleavage/polyadenylation at the alternative poly (A) site at the 3’ end of exon 4, leading  to a short, female-specific version of the Dsx protein

- In male embryos, which produce no Tra protein, exon 4 is slipped, so that exon 3 is  spliced to exon 5. Exon 5 is constitutively spliced to exon 6, which is polyadenylated at  its 3’ end – leading to a longer, male specific version of the Dsx protein

- The RNA sequence to which Tra binds in exon 4 is called exonic splicing enhancer Regulation by RNA editing

- RNA editing: the sequence of a pre-mRNA is altered; as a result, the sequence of a  mature mRNA differs from that of the exons encoding it in genomic DNA - Widespread in the mitochondria of protozoans  

- Rare in higher eukaryotes  

- APOB gene 

o This is a post-transcriptional modification that alters one or more nucleotides o The APOB gene encodes both the serum protein apolipoprotein B-100 (apoB 100), which is expressed in the liver, and apoB-48, which is expressed in  intestinal epithelial cells

o The apoB-100 has two functional domains: A N-terminal domain that associates  with lipids and a C-terminal domain that binds to LDL (Low Density Lipoprotein)  receptors on cell membrane

o In the intestine (apoB-48), the deamination by enzyme at position 6666, convert  CAA codon in exon 26 for glutamine to a UAA stop codon, leading to synthesis of  a shorter apoB-48

o Only low density lipoprotein complexes can deliver cholesterol to body tissues  ▪ Thus, only ApoB-100 delivers cholesterol to tissues

Methods: In vivo transfection assays

- Measure transcription activity to evaluate proteins believed to be transcription factors - The assay system requires two plasmids

- One plasmid contains the gene encoding the putative transcription factor (protein X) - The second plasmid contains a reporter gene and one or more binding sites for protein  X

- Both plasmids are simultaneously introduced into the cells that lack the gene encoding  protein X

- The production of reporter-gene RNA transcripts is measured; alternatively, the activity  of the encoded protein can be assayed

- If reporter-gene transcription is greater in the presence of the X-encoding plasmid than  in its absence, then the protein is an activator; if transcription is less, then it is a  repressor 

- By use of plasmids encoding a mutated or rearranged transcription factor, important  domains of the protein can be identified  

The Nuclear Pore Complex (NPC)

- The nucleus is enclosed in a nuclear envelope 

- The nucleus is separate from the cytoplasm by two membranes, which form the nuclear  envelope  

- Transport of proteins from the cytoplasmic into the nucleus and movement of  macromolecules, including mRNAs, tRNAs, and ribosomal subunits, out of the nucleus  occur through nuclear pores

- Imported nuclear proteins carry specific targeting sequences known as nuclear  localization signals (NLSs) 

- An NPC is made up of multiple copies of some 30 different proteins called nucleoporins  - Ions, small metabolites, and globular protein up to about 40kDa can diffuse passively  through the central aqueous region of the nuclear pore complex  

- However, large proteins and ribonucleoprotein complexes cannot diffuse in and out of  the nucleus  

o They are actively transported through the NPC with the assistance of soluble  transport proteins that bind macromolecules and also interact with nucleoporins  

Three types of Nucleoporins  

- Structural

o Form the scaffold of the nuclear pore, which is a rig of eightfold rotational  symmetry that traverses both membranes of the nuclear envelope creating an  annulus  

- Membrane 

o A set of seven structural nucleoporins forms a Y-shaped structure about the sie  of the ribosome  

- FG-nucleoporins 

o Contain multiple repeats of short hydrophobic sequences that are rich in  phenylalanine (F) and glycine (G) residues (FG repeats)  

Transport of mRNP to cytosol  

- mRNPs and proteins pass through the Nuclear Pore Complex

o FG domains of FG nucleoporins have an extended random-coil conformation that  forms a molecular “cloud” of continuously moving random-coil polypeptides. - Balbaians rings in insect larval salivary glands allow direct visualization of mRNP export  through NPCs

- mRNPs uncoil during their passage through NPCs and then bind to ribosomes as they  enter the cytoplasm

- This uncoiling is probably a consequence of the remodeling of mRNPs as the result of  phosphorylation of mRNP proteins by cytoplasmic kinases and the action of the RNA  helicase associated with NPC cytoplasmic filaments  

mRNAs are covered with different proteins in nucleus  

- The nuclear envelope is a double membrane that separates the nucleus from the  cytoplasm  

- mRNPs are exported, nuclear export factor 1 (NXF1) and nuclear export transport 1  (NXT1) 

- NXF1 binds nuclear mRNPs through associations with both RNA and proteins in the  mRNP complex. One of the most important of these proteins is REF (RNA export factor)  – a component of the exon junction complexes  

- Gle2, an adapter protein that reversibly binds both NXF1 and a protein in the nuclear  basket, brings nuclear mRNPs to the NPC in preparation for export  

- A protein in the cytoplasmic filaments of the NPC binds an RNA helicase (Dbp5) that  functions in the dissociation of NXF1/NXF1 and other hnRNP proteins from the mRNP as  is transported through the NPC

- Some nuclear mRNP proteins dissociate early and remain in the nucleus. Others don’t,  like NXF1/NXT1 mRNP exporter, the nuclear cap-binding complex (CBC) bound to the  5’cap, and PABPN1 bound to the poly (A) tail

- eIF4E replaces CBC in cytoplasm and PABPC1 replaces PABPN1  

Reversible phosphorylation and direction of mRNP nuclear export

- SR protein phosphorylation and dephosphorylation – directs mRNP export across the  nuclear pore complex

- Mechanism:

o Step 1: Phosphorylated (yeast) SR protein Npl3 binds nascent pre-mRNAs – functions as an adapter protein that promotes mRNP exporter binding

o Step 2: When polyadenylation is completed –

▪ It stimulates Glc7 nuclear phosphatase to dephosphorylate Npl3

▪ Dephosphorylated Npl3 – recruits NXF1/NXT1 (mRNP exporter) binding  (couple’s polyadenylation completion to export; incorrect processing  

causes RNA degradation in nucleus)

o Step 3: mRNP exporter – mRNP complex diffuses (down concentration gradient)  through the nuclear pore complex (NPC) central channel

▪ Gle2 – adapter protein that brings mRNP to NPC

o Step 4: (Now in Cytoplasm) – cytoplasmic protein kinase Sky1 phosphorylates  Npl3 (cytoplasmic localization of Sky1 ensures mRNP directional export) o Step 5: Phosphorylated Npl3 dissociates from the mRNP exporter (probably  through the action of an RNA helicase associated with NPC cytoplasmic  filaments).

o Step 6: mRNP exporter and phosphorylated Npl3 are transported back into the  nucleus through NPCs 

o Step 7: Transported mRNA is available for translation in the cytoplasm.  

Protein Synthesis

Nuclear Localization Signal (NLS)

- Nuclear transport receptors escort proteins containing NLS into the nucleus  - All proteins found in the nucleus-such as histones, transcription factors, and DNA and  RNA polymerases – are synthesized in the cytoplasm and imported into the nucleus  through the NPC

- Such proteins contain a NLS that directs their selective transport into the nucleus  - Experiment  

o Normal pyruvate kinase by itself stays in cytosol

o When a chimeric pyruvate kinase containing the SV40 NLS at its N-terminus was  expressed in cells, it was localized to the nucleus

Transport into and out of the nucleus  

- Ran is a small monomeric G protein that exists in either a GTP-bound or a GDP-bound  conformation

- It is the cycling of Ran between GTP-bound and GDP-bound conformation, leading to  the net hydrolysis of GTP to GDP, that ultimately provides the energy to drive  unidirectional transport of macromolecules through the nuclear pore

Import into of the nucleus  

- Mechanism  

o Import of cytoplasmic cargo proteins mediated by a nuclear transport receptor  known as importin

o Free importin in the cytoplasm binds to its cognate NLS in a cargo protein,  forming an importin-cargo complex

o The cargo complex then translocates through the NPC channel as the importin  interacts with FG repeats  

o The cargo complex rapidly reaches the nucleoplasm, and there the importin  interacts with Ran•GTP, which causes a conformation change that decreases the  importin’s affinity for the NLS, releasing the cargo protein into the nucleoplasm  

o The importin- Ran•GTP complex then diffuses back through the NPC  o Once the importin- Ran•GTP complex reaches the cytoplasmic side of the NPC,  Ran interacts with a specific GTPase activating protein (Ran-GAP) that is a  component of the NPC cytoplasmic filament.  

o This interaction stimulates Ran to hydrolyze it bound GTP to GDP, which causes it  to convert to a conformation that has low affinity for the importin, so that the  importin is released into the cytoplasm, where it can participate in another cycle  of import  

o Ran•GDP travels back through the pore to the nucleoplasm, where it encounters  a specific guanine nucleotide exchange factor (Ran-GEF) that causes Ran to  release its bound GDP in favor of GTP  

- The overall process of transport of cargo into the nucleus is unidirectional  - Because of the rapid dissociation of the complex when it reaches the nucleoplasm, there  is a concentration gradient of importin-cargo complex across the NPC: high in the  cytoplasm, where the complex assembles, and low in the nucleoplasm where it  dissociates  

- A similar concentration gradient is responsible for driving importin from the nucleus  back into the cytoplasm. The concentration of the importin- Ran•GTP complex is higher  in the nucleoplasm, where it assembles than on the cytoplasmic side, where it  dissociates  

- The direction of the transport processes depends on the localization of the Ran•GEF  predominately in the nucleoplasm and the Ran•GAP predominately in the cytoplasm  

Export out of the nucleus  

- Exportin 1, a nuclear transport receptor forms a complex with Ran•GTP in the nucleus  and then binds the NES (leucine rich nucleus export signal) in a cargo protein

- Binding of exportin 1 to Ran•GTP causes a conformation change in exportin 1 that increases its affinity for the NES, so that a trimolecular cargo complex if formed  - Exportin 1 interacts transiently with FG-repeats in FG-nucleoporins and diffuses through  the NPC  

- The cargo complex dissociates when it encounters the Ran•GAP associated with the  NPC cytoplasmic filaments which stimulate GTP hydrolysis, converting Ran•GTP to  Ran•GDP. This shifts it into a conformation that has low affinity for exportin 1

- After Ran•GDP dissociates from the trimolecular cargo complex, the NES-containing  cargo protein is released into the cytosol  

- Exportin 1 and Ran•GDP are transported back into the nucleus through an NPC - Ran•GEF in the nucleoplasm then stimulates conversion of Ran•GDP to Ran•GTP  

Differences between Import and Export  

- Ran•GTP is part of the cargo complex during export, but not during import  - Import – Importin, Export – Exportin  

Similarities between Import and Export  

- In the two processes, association of a nuclear transport receptor with Ran•GTP in the  nucleoplasm causes a conformational change that affects its affinity for the transport  signal  

o Import – the interaction causes release of the cargo  

o Export – the interaction promotes association with the cargo  

- In both, stimulation of Ran•GTP hydrolysis in the cytoplasm by Ran•GAP produces a  conformational change in Ran that releases the nuclear transport receptor  

Control of mRNA in cytoplasm

- The cap is usually required for translation, in addition to protection from exo ribonuclease-based degradation

- 5’UTR and 3’UTR can regulate translation

- Poly A tail is needed for mRNA stability 

Regulation of mRNA half-life

- The concentration of an mRNA is a function of both its rate of synthesis and its rate of  degradation  

- The stability of an mRNA also determines how rapidly synthesis of the encoded protein  persists ling after transcription of the gene is expressed  

- Three pathways are used to degrade mRNAs

o Deadenylation-dependent mRNA decay

o Deadenylation-independent mRNA decay  

o Endonuclease-mediated mRNA decay  

- Deadenylation-dependent mRNA decay

o Most common  

o The poly (A) tail is progressively shortened by a deadenylation complex until it  reaches a length of 20 or fewer A-residues, at which point the interaction  between PABPC1 and the remaining poly (A) is destabilized, leading to weakened  interactions between the 5’cap and translation initiation factors  

o The deadenylated mRNA then may either (1) be decapped by the DCP1/DCP2  deadenylation complex and degraded by XRN1, a 5’ -> 3’ exonuclease or (2) be  degraded by 3’-> 5’ exonucleases in cytoplasmic exosomes  

- Deadenylation-independent mRNA decay  

o Certain sequences at the 5’ end of an mRNA make the cap sensitive to the  decapping enzyme  

o These mRNAs are decapped before they are deadenylated and then degraded by  the XRN1 5’ -> 3’ exonuclease  

o An example from yeast, an RNA binding protein Rps28B binds a sequence in the  3’-UTR of its own mRNA, which tend interacts with EDc3 (enhancer of decapping  3)  

o Edc3 then recruits the DCP1/2 decapping enzyme to the mRNA, auto regulating  expression of Rps28B

- Endonuclease-mediated mRNA decay 

o Decapping or deadenylation not involved  

o Some mRNAs are cleaved internally by endonuclease and the fragments  degraded by a cytoplasmic exosome and the XRN1 exonuclease  

- The rate of mRNA deadenylation varies inversely with the frequency of translation  initiation for an mRNA: the higher the frequency of initiation, the slower the rate of  deadenylation  

- For an mRNA that is translated at a higher rate, the initiation factors are bound to the  cap much of the time, stabilizing the binding to PABPC1 and thereby protecting the poly  (A) tail from deadenylation nucleus complexes

Regulation of translation at initiation  

- Control of intracellular iron concentrations by the iron-response element-binding  protein (IRE-BP) is an example of a system in which a single protein regulates the  translation of one mRNA and the degradation of another  

- Production of Ferritin, an intracellular protein that binds and stores excess cellular iron o The 5’ UTR of ferritin mRNA contains iron-response elements (IREs) that have a  stem loop structure  

o IRE-BP recognizes five specific bases in the IRE loop and the duplex nature of the  stem  

o At low iron concentration, IRE-BP is in an active conformation that binds to the  IREs

▪ The bound IRE-BP blocks the small ribosomal subunit from scanning for  the AUG start codon, thereby inhibiting translation initiation  

▪ The resulting decrease in ferritin means that less iron is complexed with  ferritin, and therefore more iron is available to iron-requiring enzymes  

o At high iron concentration, IRE-BP is in an active conformation that does not bind  to the 5’ IREs, so translation initiation can proceed. The newly synthesized  ferritin then bends free iron ions, preventing their accumulation to harmful  levels  

- TfR mRNA - Makes transferrin receptor protein that binds and transports iron into cells o The 3’ UTR of TfR mRNA contains IREs whose stems have destabilizing AU rich  elements  

o At high iron concentration, when IRE-BP is in its active, nonbinding  

conformation, these AU-rich elements promote degradation of TfR mRNA that  leads to rapid degradation of other short-lived mRNAs with AU rich elements ▪ The resulting decrease in production of the transferrin receptors quickly  reduces iron import, thus protecting the cell from excess iron  

o At low iron concentration. IRE-BP is active and can bind to the 3’ IREs in TfR  mRNA. The bound IRE-BP blocks recognition of the AU-rich elements by the  proteins that would otherwise lead to rapid degradation of the mRNAs  

▪ As a result, production of the transferrin receptor increases, and more  iron is transported into the cell

The Translation Apparatus

- mRNA------------------------------the message  

- tRNAs------------------------------the adaptors  

- aatRNA synthetases---------------the chargers  

- amino acids------------------------the links  

- 40S ribosome---------------------the coordinator 1  

- 60S ribosome-----------------------the coordinator 2

- eIF1, eIF2, eIF3 --------------------the initiators  

- eEF1, eEF2--------------------------the elongators  

- eRF1, eEF3s-------------------------the terminators  

- GTP and ATP------------------------the juice  

- Directed by the genetic code and base pairing  

- Produced by chromosomes and transcription

Ribosome synthesis requires function and coordination of all three RNA Polymerases

- The 28S and 5.8S rRNAs associated with the large ribosomal subunit and the single 18S  rRNA of the small subunit are transcribed by RNA Pol I 

- The 5S rRNA of the large subunit is transcribed by RNA Pol III

- The mRNAs encoding the ribosomal proteins are transcribed by RNA Pol II - Approximately 80% of total RNA in rapidly growing mammalian cells is rRNA, 15% is  tRNA

Transcription of pre-rRNA/Election  

- Electron micrograph of pre-rRNA transcription units from the nucleolus (where the  synthesis and most of the processing of pre-rRNA occurs) of a frog oocyte showed that  o The pre-rRNA genes arranged in long tandem arrays separated by non transcribed spacer regions

o The human genome has about 200 rRNA genes repeated in tandem in nucleolus  

- The genomic regions corresponding to the three mature rRNAs are always arranged in 5’  -> 3’ order: 18S, 5.8S, 28S

- In all eukaryotic cells (and even in bacterial), the pre-rRNA genes codes for regions that  are removed during processing and rapidly degraded. These regions probably contribute  to proper folding of the rRNAs but are not required once that folding has occurred.  

Protein Synthesis II

Pre-tRNAs

- Cleavage and base modification occur during processing of all pre-tRNAs; some pretRNA  are also spliced during processing  

- A 5’ sequence of variable length is absent from mature tRNAs but present in all pre tRNAs  

o These extra 5’ nucleotides are present because the 5’ end of a mature tRNA is  generated by an endonucleotytic cleavage specified by the tRNA three

dimensional structure, rather than by the start site of transcription  

o The extra nucleotides are removed by ribonuclease P (RNAseP), a  

ribonucleoprotein endonuclease  

- U residues at the 3’ end of pre-tRNA are replaced with a CCA sequence  o The CCA sequence is found at the 3’ end of all tRNAs and is required for their  charging by aminoacyl-tRNA synthetases during protein synthesis  

o Function as a quality control point, since only properly folded tRNAs are  recognized by the CCA addition enzyme  

- Methyl and isopentenyl groups are added to the heterocyclic ring of purine bases, and  the 2’-OH groups in the ribose of specific residues are methylated  

- Specific uridines are converted to dihydrouridines, pseudo uridine or ribothymidine  residues

- The introns in nuclear pre-tRNAs are shorter than those in pre-mRNA and lack the  consensus splice site sequences found in pre-mRNAs

- Splicing of pre-tRNAs is catalyzed by proteins, not by RNAs

- A pre-tRNA intron is excided in one step that entails simultaneous cleavage at both ends  of the introns

- Hydrolysis of GTP and ATP is required to join the two tRNA halves generated by cleavage  on either side of the intron

- After pre-tRNAs are processed in the nucleoplasm, the mature tRNAs are transported to  the cytoplasm through nuclear pore complexes by exportin-t

Translation  

- Translation is the process by which the nucleotide sequence of an mRNA is used as a  template to join the amino acids of a polypeptide chain in the correct order - In eukaryotic cells, protein synthesis occurs in the cytoplasm, where three types of RNA  molecules come together to perform different but cooperative functions  - Messenger RNA (mRNA)  

o Carries the genetic information transcribed from DNA in a linear form o The mRNA is read in sets of three-nucleotide sequences, called codons, each of  which specifies a particular amino acid  

- Transfer RNA (tRNA)

o Key to deciphering the codons in mRNA

o Each type of amino acid has its own subset of tRNAs, which are covalently bound  to that amino acid and carry it to the growing end of a polypeptide chain when  the next codon in the mRNA calls for it

o The correct tRNA with its attached amino acid is selected at each step because  each specific tRNA molecule contains a three-nucleotide sequence, an anticodon,  that can base-pair with its complementary codon in the mRNA

o Adaptor molecules that are about 70 to 80 nucleotides

o At least one tRNA per amino acid

o Two important features  

▪ Charged by specific amino acid

▪ Recognizes and binds to codon

- Ribosomal RNA (rRNA)

o Associates with a set of proteins to form ribosome  

o Catalyze the assemble of amino acids into polypeptide chains

o Bind tRNAs and various accessory proteins necessary for protein synthesis o Composed of large and small subunit, each of which contains its own rRNA  molecule or molecules

The genetic code  

- The genetic code is read in triplets, in which every three-nucleotide sequence, or codon,  is “read” from a specified starting point in the mRNA

- 64 possible codons in the genetic code (one of four nucleotides at each of the three  positions of a codon yields 4*4*4 = 64 possible codons)

- 61 sense codons coding for 20 amino acids

- 3 termination (stop) codons  

o UAA, UGA, UAG

- 1 initiator (start) codon coding for methionine

o AUG

- The code is degenerate, a particular amino acid can be specified by multiple codons e.g.  Leucine, Serine and Arginine are each specified by 6 different codons

- The sequence of codons that runs from a specific start codon to a stop codon is called a  reading frame

- Open reading frame (ORF) 

o sequence of codons that runs from a specific start codon to a stop codon. o Predominantly, only one ORF (open reading frame) is translated into protein. - The genetic code is essentially universal 

- 3rd position of codon is least important

o 5’ -GAG- 3’

o 1 2 3

Transfer RNAs

- Translation of proteins requires both tRNAs and enzymes called aminoacyl-tRNA  synthetase

- To participate in protein synthesis, a tRNA molecule must become chemically linked to a  particular amino acid via a high energy bond, forming an aminoacyl-tRNA - The anticodon in the tRNA then base-pairs with a codon in mRNA so that the activated  amino acid can be added to the growing polypeptide chain  

- All tRNA molecules fold into a similar stem-loop arrangement that resemble a cloverleaf  when drawn in two dimensions  

- The four stems are short double helices stabilized by Watson-Crick base pairing o Acceptor stem (found in all tRNAs)

o TψCG loop

o D loop

o Anticodon loop

- Three of the four stems have loop containing seven or eight bases at their ends, while  remaining, unlooped stem contains the free 3’ and 5’ ends of the chain

- In all tRNAs, the 3’ end of the unlooped acceptor stem, to which a specific amino acid is  attached, has the sequence CCA

- Anticodon base pairs with codon on mRNA

Aminoacyl tRNA synthetases

- The process for translating nucleic acid sequences in mRNA into amino acid sequences  in proteins involves two steps 

o 1. An aminoacyl-tRNA synthetase first couples a specific amino acid, via high energy ester bond, to either the 2’ or 3’ hydroxyl of the terminal adenosine in  the corresponding tRNA

o 2. The anticodon then base-pairs with a codon in the mRNA specifying the  attached amino acid

- This happens during translation within the ribosome

How do aa tRNA (amino acid tRNA) synthetases recognize their tRNAs?

- Each of the 20 different aa tRNA synthetases recognizes one amino acid and all its  compatible or cognate tRNAs.

- Interaction of aa tRNA synthetase with anticodon loop and acceptor arm of tRNA. - Specific bases in incorrect tRNAs that are structurally similar to a cognate tRNA will  inhibit charging of the incorrect tRNA.

- Thus, recognition of the correct tRNA depends on  

o The presence of positive interactions, and  

o The absence of negative interactions.

- aa tRNA synthetase has proofreading activity

Codon:Anticodon recognition

- (mRNA): CODON 5’ ACG 3’ (5 is the 1st one)

- (tRNA): ANTICODON 3’ UGC 5 (3 is the 1st one)

- Third base degeneracy in genetic code. Third base of the codon is a) irrelevant or b)  where only purines (A and G) and pyrimidines (U and C) are distinguished from one  another

- Often, one tRNA can recognize more than one codon. “Wobbling” may occur at third  position.

- “Wobble” rules: At third position of the codon, G=U base pairing is allowed, in addition  to normal G=C and A=U base pairing. Inosine (I) in tRNA can base pair with A, C or U. - For example, Asn tRNA has anticodon 5’ GUU 3’. It can base pair with normal AAC codon  as well as with AAU codon

Base pairing at codon:anticodon

- Wobble hypothesis

o The 1st position in tRNA anticodon (5’-most nucleotide on each anticodon),  which binds to the 3rd position in the mRNA codon (3’-most nucleotide in each  codon), can participate in non-standard pairing

- Wobble pairing allows a tRNA to recognize more than one mRNA codon; conversely, it  allows a codon to be recognized by more than one kind of tRNA

- Wobble rules: At third (wobble) position in mRNA codon,  

o G-U base pairing is allowed.  

o A, C or U can pair with Inosine (I) located in 1st position of anti-codon in tRNA. - Broader Implications 

o A single kind of tRNA may recognize more than one codon corresponding to a  specific amino acid.

- Implications 

- Third nucleotide degeneracy in genetic code. Third nucleotide of codon in mRNA is:  o Least informative

o May be distinguishable at the level of only purines (A and G) and pyrimidines (U  and C) and this too only when Inosine is not at anticodon position 1

- Side note: A in #1 of anticodon is very rare

Ribosomes

- 2 subunits (small and large subunits)

o 30S (small) and 50S (large) in prokaryotes, 40S (small) and 60S (large) in eukaryotes

- RNA-protein complexes

- Functions

o Coordinate translation and catalyze peptide bond formation

- Peptide gets transferred to amino acyl tRNA

- New peptide bond forms and chain grows by one amino acid (aa)

- Structure of ribosomes – there are binding sites for 3 tRNAs in ribosomes: o A site for aatRNA

o P site for peptidyl tRNA and  

o E site for exit tRNA

Challenges in Translation Initiation and Elongation

- Get the message to the translating “machine”, i.e. Bring mRNA and ribosome subunits  together along with other factors.

- Identify and begin reading at the correct “first word”, i.e. Locate the first readable  “AUG” codon in mRNA.

- Recruit the correct translator or “bridge” molecule over the first word, i.e. Recruit the  initiator methionine tRNA to site “P” over the first AUG codon in mRNA. - Recruit the next correct translator or “bridge” molecule over the next word, i.e. Place an  aminoacyl tRNA with anticodon complementary to the next codon in mRNA. - Connect words, i.e. Form a peptide bond between the terminal amino acid within the  growing peptide that is located on the peptidyl tRNA and the amino acid in the  aminoacyl tRNA.

- Move the machine just enough distance to be over the next word, i.e. Translocate the  ribosome over a distance of precisely one codon.

“Initiation” of protein synthesis (translation) in eukaryotes

- Step 1: An elF2 ternary complex forms when elF2•GTP binds a tRNA (Met) - Step 2: When a ribosome dissociates at the termination of translation, the 40S subunit is  bound by elF1, elFA and elF3  

o A 43S preinitiation complex forms when this subunit associates with an elF2  ternary complex and elF5  

- Step 3: an mRNA is activated when a multisubunit elF4 complex binds; elF4E binds to  the 5’ cap, and elF4G binds PABPC

o Then elF4B, which stimulates elF4A helicase activity, also joins this circular  complex in which both the mRNA 5’ cap and poly (A) tail are associated with the  elF4 complex

- Step 4: The 43S preinitiation complex binds an elF4-mRNA complex  

- Step 5: The RNA helicase activity of subunit elF4A unwinds any RNA secondary structure  at the 5’ end of the mRNA as the 40S subunit scans in the 5’ -> 3’ direction until it  recognizes the initiation codon  

- Step 6: recognition of the initiation codon causes elF5 to stimulate hydrolysis of elF2- bound GTP

o This switches the conformation of the scanning complex to a 48S initiation  complex with the anticodon of tRNA (Met) base-paired to the initiator AUG in  the 40S-subunit P site

- Step 7: the 60S subunits joins the 40S subunit, leading to the release of most of the  earlier-acting elFs as elF5B-GTP binds to elF1A in the ribosomal A site

o The release elF4 complex and elF4B associate with the 5’ cap and PABPC to  prepare for interaction with another 43S preinitiation complex

- Step 8: correct association of the 40S and 60S subunits results in hydrolysis of elF5B bound GTP, release of elF5B-GDP and elF1A, and formation of the 80S initiation complex  with tRNA (Met) base-paired to the initiation codon in the ribosomal P site

Protein synthesis: Elongation

- Step 1: Once the 80S ribosome with Met-tRNA in the ribosome P site is assembled,  aatRNA enters the A site of the ribosome

o The tRNA is accompanied by EF1a-GTP

o Many tRNAs can diffuse into the “A” site, but only those that can pair correctly  with the codon can trigger the next step

- Step 2: Hydrolysis of GTP in EF1α•GTP occurs, which causes a conformational change in  the ribosome (inducing a better fit of aatRNA in A site)

- Step 3: Formation of the new peptide bond (peptidyl transferase reaction, catalyzed by  large rRNA; peptidyl tRNA 3’ terminal “A” (from CCA) also participates in the reaction

- Step 4: EF2-GTP hydrolysis to EF2-GDP, which causes a conformational change in the  ribosome that translocates precisely one codon distance over the mRNA (and relative to  the mRNA bound tRNAs) and shifts the unacylated tRNA (Met) to the E site and the  tRNA with the bound peptide to the P site  

- Step 5: The cycle can begin again, ready for entry of next aatRNA at A site Termination of translation

- When a ribosome bearing a nascent protein chain reaches a stop codon, release factor  eRF1 enters the A site together with eRF3•GTP 

- eRF3-GTP hydrolysis to eRF3-GDP is coupled to release of C-terminal end of polypeptide,  forming a post-termination complex  

- The ribosomal subunits are separated by the action of the ABCE1 ATPase together with  eIF1, eIF1A, and eIF3

- The 40S subunit is released bound to these elFs, ready to initiate another cycle of  translation  

Ribosomes are rapidly reused in translation

- During translation, eukaryotic mRNAs are made circular by interaction of specific  proteins to enhance ribosome recycling and increase efficiency of protein synthesis. - When a ribosome completes translation and dissociates from the 3’ end, the separated  subunits can rapidly find the nearby 5’ cap and PABPC-bound poly(A) tail and initiate  another round of synthesis  

ER Membrane Proteins

Polyadenylation at the 3’ UTR of mRNA can regulate translation

- Translationally dormant 

o In immature oocytes, mRNAs containing the U-rich cytoplasmic polyadenylation  element (CPE) have short poly (A) tails  

o CPE-binding protein (CPEB) bound to the U-rich CPE interacts with the protein  Maskin, which in turn bonds to the eIF4E associated with the mRNA 5’ cap o As a result, eIF4E cannot interact with the other initiator factors or the small  ribosomal subunit, so translation initiation is blocked

- Translationally active 

o Hormone stimulation of oocytes activates a protein kinase that phosphorylates  CPEB, causing it to release Maskin

o The cleavage and polyadenylation specificity factor (CPSF) then binds to the poly  (A) tail site, interacting with the bound CPEB and the cytoplasmic form of poly  (A) polymerase (PAP)

o After the poly (A) tail is lengthened, multiple copies of cytoplasmic poly (A)- binding protein 1 (PABPC1) can bind to it and interact with elF4G, which  functions with other initiation factors to bind the 40S ribosomal subunit and  initiate translation  

Entry of ribosomes in prokaryotic mRNAs

- Prokaryotic mRNAs are polycistronic because ribosomes can bind internally to initiate  translation

- Shine-Dalgarno model of initiation

Sorting of proteins in eukaryotic cells

- Proteins are made on the ER or in cytosol and then transported to various places in the  cell

Targeting sequences direct proteins to the proper site

- Synthesis of secreted proteins, integral plasma-membrane proteins, and proteins  destined for the ER, Golgi complex, plasma membrane, or lysosome begins on cytosolic  ribosomes

Target Organelle

Location of sequence within  proteins

Removal of sequence

Endoplasmic reticulum  

(lumen)

N-terminus

Yes

Mitochondrion (matrix)

N-terminus

Yes

Chloroplast (stroma)

N-terminus

Yes

Peroxisome (matrix)

C-terminus (most proteins) N-terminus (few proteins)

No

Nucleus (nucleoplasm)

Varies

No

Endoplasmic reticulum (ER)  

- All eukaryotic cells have an endoplasmic reticulum (ER)

- A convoluted organelle, made up of tubules and flattened sacs, whose membrane is  continuous with the membrane of the nucleus  

- Usually have a large surface area

- Lipids are synthesized on smooth ER membranes

- Membrane proteins and secreted proteins are made on rough ER and are first delivered  into ER lumen

Secretory proteins enter the ER lumen

- Pulse-chase experiments with purified ER membranes: demonstrate that secretory  proteins are localized in the ER lumen shortly after synthesis

- Experiment:

o Cells are incubated for a brief time with radiolabeled amino acids – only newly  synthesized proteins become labeled

o Cells homogenized – fractures the plasma membrane and shears the rough ER  into small vesicles called microsomes

o Microsomes with bound ribosomes – isolated by differential and sucrose  density-gradient centrifugation  

o Purified microsomes treated with a protease – results

o (–) Detergent – proteins in ER are protected from digestion

o (+) Detergent (control) – dissolves ER membrane – proteins no longer protected  from digestion

- Conclusion: newly made proteins are inside the microsomes (lumen of the rough ER)  after synthesis.

Ribosomes can be localized on the ER!

- A signal sequence, SRP, and SRP receptor system docks the ribosome on an ER  translocon and co-translationally inserts the nascent protein into or through the ER  membrane.  

- Membrane-bound ribosomes – recruited to the ER during synthesis of a polypeptide  containing an ER signal sequence

- Free cytosolic ribosomes and ER membrane-bound ribosomes are identical Experiments to prove that translation and translocation occur simultaneously

- Cell-free experiments demonstrate that translocation of secretory protein into  microsomes (equivalent to ER membrane) is coupled to translation

o Treatment of microsomes with EDTA, which chelates Mg2+ ions, strips them of  associated ribosomes, allowing isolation of ribosome-free microsomes, which  are equivalent to the ER membrane  

- Protein is found in microsomes (i.e. translocated) only if microsomes are present  DURING protein synthesis. Protein (mature) has no signal peptide sequence o Decrease in molecular weight of protein

The signal recognition particle (SRP)

- A cytosolic ribonucleoprotein particle that transiently binds to both the ER signal  sequence in a nascent protein and large (60S) ribosomal subunit, forming a large  complex

- Composed of 6 proteins bound to a 300 nucleotide RNA; functions as a scaffold for  proteins

- P54 – binds ER signal sequence

o Can be chemically cross-linked to ER signal sequences, which shows that thus  subunit is the one that binds to the signal sequence in a nascent secretory  protein  

o A region of P54 known as the M domain, containing many methionine and other  amino acid residues with hydrophobic side chains, contains a cleft whose inner  surface is lined by hydrophobic side chains

- P68/P72 – required for protein translocation  

- P9/P14 – interact with ribosomes  

- The bacteria Ffh protein is homologous to the portion of P54 that binds ER signal  sequences  

The SRP and SRP receptor need GTP to bind to one another

- Each protein has one bound GTP. When the SRP binds to its receptor on the ER surface,  the two GTPs interact with one another and contribute to the binding. When the GTPs  are hydrolyzed, the proteins dissociate

- Neither subunit alone contains a complete active site for the hydrolysis of GTP; only  when they interact, can they form a complete active site for GTP hydrolysis

Co-translational translocation

- Once the ER signal sequence emerges from the ribosome, it is bound by a SRP - The SRP and the nascent polypeptide chain-ribose complex bind to the SRP receptor in  the ER membrane. This interaction is strengthened by the binding of GTP to both the  SRP and its receptor  

- Transfer of the nascent polypeptide-ribosome to the translocon (a complex of proteins  that forms a channel embedded within the ER membrane) leads to opening of this  translocation channel to admit the growing polypeptide adjacent to the signal sequence,  which is transferred to a hydrophobic binding site next to the central pore

- Both the SRP and SRP receptor, once dissociated from the translocon, hydrolyze their  bound GTP and then are ready to initiate the insertion of another polypeptide chain - As the polypeptide chain elongates, it passes through the translocon channel into the ER  lumen, where the signal sequence is cleaved by signal peptidase as is rapidly degraded

- The peptide chain continues to elongate as the mRNA is translated toward the 3’ end.  Because the ribosome is attached to the translocon, the growing chain is extruded  through the translocon into the ER lumen

o N-linked glycosylation

- Once translation is complete, the ribosome is released, the remainder of the protein is  drawn into the ER lumen, the translocon closes, and the protein assumes its native  folded conformation  

o Disulfide bonds form and protein folds in lumen

Post-translational translocation in yeast

- In most eukaryotes, secretory proteins enter the ER by co-translational translocation. In  yeast, however, some secretory proteins enter the ER lumen after translation has been  completed  

- SRP and SRP receptor are not involved in post-translational translocation  - The driving force for unidirectional translocation across the ER membrane is provided by  an additional protein complex known as the Sec63 complex and a member of the Hsp70  family of molecular chaperones known as BiP 

- Sec63 is embedded in the ER membrane in the vicinity of the translocon, whereas BiP is  within the ER lumen

- BiP has a peptide-binding domain and an ATPase domain.  

- These chaperones bind and stabilize unfolded or partially folded proteins  - Mechanism  

o Once the N-terminal segment of the protein enters the ER lumen, signal  peptidase cleaves the signal sequence just as in co-translational translocation o Interaction of BiP•ATP with the luminal portion of the Sec63 complex causes  hydrolysis of the bound ATP, producing a conformational change in BiP that  promotes its binding to an exposed polypeptide chain  

o Binding of a molecule of BiP•ADP to the luminal portion of the polypeptide  prevents backsliding of the polypeptide out of the ER

o Successful binding of BiP•ADP molecules to the polypeptide chain acts as a  ratchet, ultimately drawing the entire polypeptide into the ER within a few  seconds  

o The BiP molecules spontaneously exchange their bound ADP for ATP, leading to  release of the polypeptide, which can then fold into its native conformation  o The recycled BiP•ATP is then ready for another interaction with Sec63

Structure of the translocon

- Sec61α, β and ƴ makes up channel

- The translocon was first identified through mutations in the yeast gene encoding a  protein called Sec61α

- Three proteins called Sec61 complex were found to form the mammalian translocon

- Cross-linking experiments show that Sec61α is a translocon component that contacts  nascent secretory proteins as they pass into the ER lumen

- The large ribosomal subunit is aligned with the pore of the translocon in such a way that  the growing chain is never exposed to the cytoplasm and is prevented from folding until  it reaches the ER lumen

Bacterial Sec61 complex

- Structure explains how peptide chain is threaded through translocon and how it  emerges laterally into membrane.

o The Sec61 complex shows how the translocon preserves the integrity of the  membrane  

- Isoleucine residues at the constricted waist of the pore forms a gasket that keeps the  channel sealed to small molecules even as a translocating polypeptide passes through  the channel

- When no translocating peptide is present, the channel is closed by a short helical plug. o This plug moves out of the channel during translocation

- Helices can separate like “opening a gate” to allow lateral passage of a hydrophobic  transmembrane domain into the lipid bilayer  

Several Topological classes of integral membrane proteins are synthesized on the ER

- The topology of a membrane protein refers to the number of times its polypeptide chain  spans the membrane and the orientation of those membrane-spanning segments within  the membrane  

- The key elements of a protein that determine its topology are the membrane-spanning  segments themselves, which are usually α helices containing 20-25 hydrophobic amino  acids that contribute to energetically favorable interactions within the hydrophobic  interior of the phospholipid bilayer  

- The topological classes of integral membranes proteins are synthesized on the rough ER o Class I, II, III, the tail-anchored proteins and IV

- Class I, II, III, and the tail-anchored proteins are single-pass membrane proteins  o Have only one membrane-spanning α-helical segment  

- Type I proteins  

o Possess an internal hydrophobic sequence of approximately 22 amino acids that  becomes the membrane-spanning α helix

o Cleaved N-terminal ER signal sequence  

o Anchored in the membrane with their hydrophilic N-terminal region on the  luminal face (exoplasmic face) and their hydrophilic C-terminal region on the  cytosolic face

- Type II proteins

o No cleaved ER signal sequence  

o Oriented with their hydrophilic N-terminal region on the cytosolic face and their  hydrophilic C-terminal region on the luminal face

- Type III proteins  

o No cleaved ER signal sequence  

o Have hydrophobic membrane-spanning segment at their N-terminus and thus  have the same orientation as type I proteins

- Tail-anchored proteins

o Have a hydrophobic segment at their C-terminus that spans the membrane  - Type IV  

o Contain two or more membrane-spanning segments and are sometimes called  multipass membrane proteins  

Proteins can be inserted in the ER membrane a number of ways

- Exoplasmic space Lumen of ER, Golgi and exterior of cell

- The final orientation of membrane proteins is established during their biosynthesis on  the ER membrane

Insertion of single pass proteins with Stop-transfer-anchor signal sequence

- Type I proteins 

- The N-terminal signal sequence of a nascent type I protein initiates co-translational  translocation of the protein through the combined action of the SRP and SRP receptor  - It is called stop-transfer anchor sequence because it functions both to stop passage of  the polypeptide chain through the translocon and to become a hydrophobic  transmembrane segment in the membrane bilayer  

- Mechanism  

o Step 1: After, the nascent polypeptide chain-ribosome complex becomes  associated with a translocon in the ER membrane, the N-terminal signal  sequence is cleaved  

o Step 2-3: the chain is elongated until the hydrophobic stop-transfer anchor  sequence is synthesized and enters the translocon, where it prevents the  nascent chain from extruding farther into the ER lumen

o Step 4: The stop-transfer anchor sequence moves laterally through a  hydrophobic cleft between translocon subunits and ultimately becomes  anchored in the phospholipid bilayer  

o Step 5: As synthesis continues, the elongating chain may loop out into the  cytosol through the small space between the ribosome and translocon

o Step 6: when synthesis is complete, the ribosomal subunits are released into the  cytosol, leaving the protein free to diffuse laterally in the membrane  

Insertion of single-pass protein with internal Signal-anchor sequence

- Type II and III 

- Both possess a single internal hydrophobic signal anchor sequence that functions as  both an ER signal sequence and a membrane anchor  

- Type II

o The internal signal anchor sequence in type II proteins directs insertion of the  nascent polypeptide chain into the ER membrane so that the N-terminus of the  chain faces the cytosol

o The signal anchor sequence is not cleaved and eventually moves laterally from  the signal-sequence binding site at the edge of the translocon directly into the  phospholipid bilayer, where it functions as a membrane anchor  

o As elongation continues, the C-terminus region of the growing chain is extruded  through the translocon into the ER lumen by co-translational translocation  o Basic amino acids (with +ve charge) BEFORE (located N’ to) the internal signal  sequence

- Type III

o The signal anchor sequence, which is located near the N-terminus, directs  insertion of the nascent chain into the ER membrane with its N-terminus facing  the lumen, in an orientation opposite to that of the signal anchor in type II

o The signal anchor also functions like a stop-transfer sequence and prevents  further extrusion of the elongating chain into the ER lumen

o Continued elongation of the chain C-terminal to the signal anchor sequence  proceeds as it does for type I proteins  

o Basic amino acids (with +ve charge) AFTER (located C’ to) the internal signal  sequence

How proteins become anchored with GPI (Glycosyl Phosphatidyl inositol)

- Some cell-surface proteins are anchored to the phospholipid bilayer not by a sequence  of hydrophobic amino acids, but by a covalently attached amphipathic molecule,  glycosylphosphatidylinositol (GPI)

- GPI transamidase cleaves protein and transfers the carboxyl group of the new C terminus to the terminal amino group of a preformed GPI in ER membrane - Protein is first inserted as single pass membrane protein (type I).

- Proteins with GPI anchor can diffuse relatively fast in the plane of the bilayer.

Membrane transport

Composition of a membrane

- Fluid mosaic model of biomembranes.  

- Plasma membrane – defines the cell and separates the cytosol from the extracellular  environment  

- Bio-membranes have the same basic architecture: phospholipid bilayer (~3 nm wide)  with embedded proteins.

- According to the fluid mosaic model of bio-membranes the phospholipid bilayer behaves in some respect like a 2D fluid

- Fluidity: (similar to that of olive oil)

o Noncovalent interactions between phospholipids, and between phospholipids  and proteins provide membrane integrity and resilience.  

o Individual phospholipids spin and diffuse laterally within the plane of the  membrane.

- Barrier: hydrophobic core of the bilayer prevents unassisted movement of water soluble substances from one side to the other.  

- Proteins: membrane proteins provide each cellular membrane its unique set of  functions.  

o Integral membrane proteins (transmembrane proteins) span the bilayer and  often form dimers and higher-order oligomers.

o Lipid-anchored proteins – tethered to one leaflet by a covalently attached  hydrocarbon chain  

o Peripheral proteins – associate primarily by specific noncovalent interactions  with integral membrane proteins or membrane lipids [include cytoskeletal  proteins]

- Proteins in the plasma membrane also make extensive contact with the cytoskeleton Phospholipids

- Serves as a permeability barrier, helping to maintain the characteristics differences  between the inside and the outside of the cell or organelle  

- Amphipathic molecules  

o A fatty acid-based (fatty acyl) hydrocarbon “tail” that is hydrophobic o A polar head group that is hydrophilic  

- Phospholipids can aggregate into three structures  

o Spherical micelles  

o Liposomes  

o Sheet like phospholipid bilayer  

Three classes of membrane lipids

- Phosphoglycerides

o Most common and abundant class of phospholipids  

o Consists of a hydrophobic tail composed of two fatty acid-based (acyl) chains  esterified to the two hydroxyl groups in glycerol phosphate and a polar head group attached to the phosphate group

▪ The two fatty acyl chains may differ in the number of carbons that they  contain and their degree of saturation  

o A Phosphoglyceride is classified according to the nature of its head group  o Plasmalogens are a group of phosphoglycerides that contain one fatty acyl chain  attached to glycerol by an ester linkage and one attached by an ether linkage;  they contain the same head groups as other phosphoglycerides  

▪ Abundant in the human brain and heart tissue  

- Sphingolipids

o Derivatives of sphingosine, an amino alcohol with a long hydrocarbon chain o Various fatty acyl chains are connected to sphingosine by an amide bond  - Sterols

o A four-ring hydrocarbon  

o Amphipathic  

o The hydroxyl group is equivalent to the other polar head group in other lipids;  the conjugated ring and short hydrocarbon chain from the hydrophobic tail  

Proteins in membranes

- Integral membrane proteins (have one or more alpha helical or beta sheet  transmembrane motif)  

- Lipid anchored proteins (are covalently attached to a lipid)

- Peripheral membrane proteins (bound to polar heads or other proteins Cell membranes are two-faced

- Cytoplasmic face and exoplasmic face

- Exoplasmic  

o Plasma membrane -> Directed away from the cytosol, toward the extracellular  space or external environment, and defines that outer limit of the cell

- Cytosolic face

o Faces the cytosol

- Plasma membrane: a single bilayer that encloses the cell  

o Cytosolic and exoplasmic leaflets/faces of the bilayer

- Vesicles and some organelles:  

o Bounded by single membranes.

o Internal aqueous space – topologically equivalent to the outside of the cell  - Nucleus, mitochondrion, and chloroplast (not shown) organelles:

o Enclosed by two membranes separated by a small intermembrane space

o Exoplasmic faces of the inner and outer membranes border the intermembrane  space

- Endocytosis:

o Plasma membrane segment – buds inward toward the cytosol and eventually  pinches off a separate vesicle  

▪ Cytosolic face – remains facing the cytosol  

▪ Exoplasmic face – faces vesicle lumen  

- Exocytosis:

o An intracellular vesicle fuses with the plasma membrane.

▪ Vesicle lumen connects with the extracellular medium.  

▪ Cytoplasmic face remains facing cytoplasm.

- Membrane-spanning proteins retain asymmetric orientation during vesicle budding and  fusion; same protein segment(s) always faces the cytosol.

Transport from one compartment to another

- Transport of membrane and soluble proteins from one membrane-bounded  compartment to another is mediated by transport vesicles that collect cargo proteins in  buds arising from the membrane of one compartment and then deliver these cargo  proteins to the next compartment by fusing with the membrane of that compartment

- New vesicles form by BUDDING from donor compartment and undergo FUSION with  target compartment

Intracellular transport involves budding and fusing of vesicles

- The budding of a vesicle from its parent membrane is driven by the polymerization of  soluble protein complexes on the membrane to form a proteinaceous vesicle coat  - The coat gives curvature to the membrane to form a vesicle and acts as the filter to  determine which proteins are admitted into the vesicle  

- Three types of vesicles

o COPII – ER to c-Golgi

o COPI – between Golgi, c-Golgi to ER

o Clathrin – from plasma membrane and the trans-Golgi to endosomes - Budding is initiated with small G protein (ARF, Sar1

o ARF

▪ COPI

▪ Clathrin  

o Sar1

▪ COPII

- Complexes of coat proteins in the cytosol then bind to the cytosolic domain of  membrane cargo proteins, some of which also act as receptors that bind soluble  proteins in the lumen, thereby recruiting luminal cargo proteins into the budding vesicle

- After being released and shedding its coat, a vesicle fuses with its target membrane in a  process that involves interaction of cognate SNARE proteins (v-SNARES and t-SNARES)

A coat assembles over budding vesicles: Role of Sar1 in COPII transport

- Step 1: Interaction of soluble GDP-bound Sar1 with the GEF Sec12, an ER membrane  protein, catalyzes exchange of GTP for GDP on Sar1

o The hydrophobic N-terminus of the GTP-bound form of Sar1 extends outward  from the protein’s surface and anchors Sar1 to the ER membrane

- Step 2: The membrane-attached Sar1•GTP drives the polymerization of cytosolic  complexes of COPII subunits on the membrane, eventually leading to formation of  vesicle buds  

- Step 3: Once COPII vesicles are released from the donor membrane, the Sar1 GTPase  activity hydrolyzes Sar1•GTP in the vesicle membrane to Sar1•GDP with the assistance  of one of the coat subunits (Sec23 and Sec24)

o Sec23 is a GAP for Sar1

- Step 4: This hydrolysis trigger disassembly of the COPII coat  

Docking and fusion; role of G proteins and SNARES

- A second set of small GTP-binding proteins, known as Rab proteins, associate with  transport vesicles and act as key regulators of vesicle trafficking to and fusion with the  appropriate target membrane  

- Rab belong to the GTPase superfamily of switch proteins  

- Mechanism

o Step 1: A Rab protein tethered via a lipid anchor to a secretory vesicle binds to  an effector protein complex on the plasma membrane, thereby docking the  transport vesicle on the appropriate target membrane

o Step 2: A v-snare, VAMP (Vesicle Associated Membrane Protein) interacts with  the cytosolic domains of the cognate t-SNAREs (syntaxin and SNAP 25)  

▪ The very stable coiled coil SNARE complexes that are formed hold the  vesicle close to the target membrane  

o Step 3: Fusion of the two membranes immediately follows formation of SNARE  complexes

o Step 4: NSF in conjunction with α-SNAP binds to the SNARES complexes  ▪ The NSF-catalyzed hydrolysis of ATP then drives dissociation of the SNARE  complexes, freeing the SNARE proteins for another round of vesicle  

fusion

▪ Also at this time, Rab•GTP is hydrolyzed to Rab•GDP and dissociates from  the Rab effector

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