Essay: ER translocon

The first step of translation is called initiation. In this step, mRNA, a tRNA containing the first amino acid of the polypeptide, and two ribosomal subunits come together to start the process. The small subunit then binds to both mRNA and a specific initiator tRNA, which contains the amino acid methionine (MET). Next, the subunit scans along the mRNA strand until it reaches the start codon AUG which indicates the start of translation process. The start codon also establishes the reading frame for the mRNA strand, with is crucial to synthesizing the protein. Then, the tRNA initiator then binds to the start codon via hydrogen bonding. The complex of consisting of mRNA, initiator tRNA, and the small ribosomal subunit attaches to the large ribosomal subunit, which completes the initiation complex.
These components are brought together by the help of proteins called initiation factors which bind to the small ribosomal subunit during initiation. Once the formation of the initiation complex is complete, the initiator tRNA attaches to the P site of the ribosome, and the empty A site is ready for the next aminoacyl tRNA. The polypeptide is always synthesized in one direction, which is from the N-terminus to the C-terminus direction.
The mechanisms whereby ribosomes engage a messenger RNA and select the start site for translation differ between prokaryotes and eukaryotes. Initiation sites in prokaryotic mRNAs are usually selected via base pairing with 16S ribosomal RNA (rRNA) in the small (30S) ribosomal subunit with the specific Shine–Dalgarno sequence in mRNA. These sequence are found before the start codons (AUG codon) and directs them to the ribosomal P site.
On the other hand, in eukaryotes, most mRNAs are translated by a scanning mechanism wherein the small (40S) ribosomal subunit is preloaded with Met-tRNAi by the GTP-bound form of eukaryotic initiation factor 2 (eIF2)—a GTPase in a reaction promoted by eIF1, -1A, and -5 and the multi-subunit eIF3. the resulting 43S preinitiation complex (PIC) then attaches to the 5’ end of the mRNA and scans along in the 3’ direction and stopping at the start codon (often AUG). Eukaryotes initiation also differs from prokaryotes in that, its scanning mechanism is limited by the inclusion of stable stem-loop structures in the 5’ UTR upstream of the start codon as well as the fact that there is a m7G cap at the 5’ end of mRNA and the cap- binding complex eIF4F (which attaches to the cap to activate mRNA for 43S PIC attachment) which acts to facilitate the scanning process.
(b). What is the role of the PIC in Initiation of protein synthesis?
The 43S preinitiation complex is an important intermediate complex during cap-dependent translation initiation. In translation initiation, the 43S PIC is pre-formed as a stable complex and recruited to the five-prime cap of eukaryotic messenger RNAs (mRNAs) by the eIF4F complex. The PIC employs an intricate molecular network to communicate start codon recognition and trigger the transition to its closed, scanning-arrested state.
Initiation begins with the formation of the ternary complex (TC) containing eIF2-GTP and the initiator tRNA. The ternary complex is recruited to the 40S subunit with the help of eIFs 1, 1A, 3 and 5 to form the PIC . Meanwhile, the mRNA is bound by the eIF4 factors and the PABP to form an activated mRNP, which is then recruited to the PIC. Once bound at the 5′ end of the mRNA, the PIC scans to locate the start (AUG) codon. Start codon recognition triggers eIF1 release and conversion of eIF2 to its GDP-bound state, arresting the scanning process. eIF2-GDP and eIF5 dissociate, clearing the way for eIF5B to mediate joining of the 60S subunit. Subunit joining is followed by GTP hydrolysis by eIF5B and factor dissociation to form the 80S initiation complex (IC).
The 43S preinitiation complex is an important intermediate complex during cap-dependent translation initiation. In translation initiation, the 43S PIC is pre-formed as a stable complex and recruited to the Five-prime cap of eukaryotic messenger RNAs (mRNAs) by the eIF4F complex. The 43S PIC then scans in the 5′ – 3′ direction along the mRNA in an ATP-dependent fashion to locate the start codon. Start codon recognition occurs through base-pairing between the Met-tRNAiMet and AUG in the ribosomal P-site and a number of associated changes, and is followed by joining of the large 60S ribosomal subunit to form the 80S ribosome that is ready to begin peptide synthesis – elongation phase.
(c). What is the cap binding protein and what is its relationship to the PIC?
The cap binding protein, eIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of the eIF4F pre-initiation complex. Almost all cellular mRNA require eIF4E in order to be translated into protein. The eIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. A cap binding protein makes up a cap-binding complex which acts to facilitate the scanning mechanism. The eIF4F (cap-binding complex) is made up of the cap-binding protein eIF4E, eIF4G, and the RNA helicase eIF4A. Binding of the cap by eIF4E is often considered the rate-limiting step translation initiation in the cytoplasm and the concentration of eIF4E is a regulatory within translational control. The cap binding protein attaching to the cap to activate mRNA for it to be attached to the 43S preinitiation complex.
(d). There are numerous Initiation factors, Elongation and Termination factors. In general terms what are there roles?
Each stage of translation—initiation, chain elongation, and termination—requires specific protein factors, including GTP-binding proteins that hydrolyze their bound GTP to GDP when a step has been completed successfully. Translation initiation in eukaryotes requires the involvement of multiple initiation factors (eIFs). Initiation factors are a specific group of proteins that mediate the assemble 40 S ribosomal subunit around an mRNA that specifically has a Met-tRNAiMet correctly positioned at the start codon in the ribosomal P site. This is especially needed for eukaryotes due to the fact that unlike prokaryotes, they lack in their Shine- Dalgarno sequence, the 16 S rRNA that allows it to bind in close proximity to an initiation codon.
Initiation factor eIF4F composed of eIF4E, eIF4A, and eIF4G, binds to the 5’-cap structure of an mRNA and prepares an mRNA for recruitment of a 40 S subunit. Initiation factor eIF4G which is composed within eukaryotic initiation factor 4F (IF4F) assists in recruiting the 40S ribosomal subunit to mRNA described above. Initiation factor eIF4B supports ATP-dependent RNA helicase activity of eIF4F AND eIF4A needed to unravel the secondary structure present. In addition to this, numerous initiation factors act to bind GTP as the hydrolysis of GTP to GDP acts as a barrier which allows consequent steps to proceed if the previous step has been carried out correctly.
The cap structure is bound by translation initiation factor 4F (eIF4F) as the first step of cap-dependent translation. eIF4F consists of three subunits, eIF4E, eIF4A, and eIF4G. eIF4E binds the cap structure directly and consequently is required for cap-dependent translation.
Translational elongation factors are proteins that play two important roles during the elongation cycle of protein biosynthesis on the ribosome. First, elongation factors are involved in bringing aminoacyl-tRNA (aa-tRNA) to the ribosome during protein synthesis. Second, an elongation factor is involved in translocation, the step in elongation at which the peptidyl-tRNA is moved from one ribosomal site to another as the mRNA moves through the ribosome.
There are 2 types of termination factors in translation. One of the factors acts to recognize stop codons while the one acts to promote hydrolysis of peptidyl-tRNA.
Parker, J. “Elongation Factors; Translation.” Encyclopedia of Genetics, 2001, pp. 610–611., doi:10.1006/rwgn.2001.0402
Hinnebusch, Alan G. “The Scanning Mechanism of Eukaryotic Translation Initiation.” Annual Review of Biochemistry, vol. 83, no. 1, Feb. 2014, pp. 779–812., doi:10.1146/annurev-biochem-060713-035802.
Gallie, D. R. (2014). The role of the poly(A) binding protein in the assembly of the Cap-binding complex during translation initiation in plants. Translation, 2(2), e959378. http://doi.org/10.4161/2169074X.2014.959378
Kozak, Marilyn. “Initiation of translation in prokaryotes and eukaryotes.” Gene, vol. 234, no. 2, 1999, pp. 187–208., doi:10.1016/s0378-1119(99)00210-3.
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 4.5, Stepwise Formation of Proteins on Ribosomes. Available from: https://www.ncbi.nlm.nih.gov/books/NBK21653/
2. Describe the structure and function of the SRP. (a)How does the SRP inhibit translation?
Translation is inhibited by SRP while it transport the incomplete polypeptide to the peptide translocation complex. Firstly, the ribosome which begins to synthesis the protein recognizes mRNA. As the peptide chain is synthesized, its (the amino terminus (the starting segment) containing its signal sequence proteins are recognized by SPR. SPR binding to the signal sequence as well as the ribosome causes translation to come to a halt (inhibition). SRP’s function is to guide the ribosome and the growing polypeptide to SRP’S receptors in the cytosolic side of the endoplasmic reticulum. This incomplete polypeptide is then transported to the peptide translocation complex in the ER. After this, the SPR dissociates from the ribosome an the polypeptide synthesis continues. (Alberts)
(b)How do the SRP and the ribosome find their place on the ER? Describe docking.
During translation, a signal sequence is synthesized. As the signal peptide departs from the ribosome, it binds to the signal recognition particle. The signal recognition particle (SRP) can be described a huge protein complex which quickly binds to the signal peptide as it is being synthesized and the ribosome. SRP’s function is to guide the ribosome and the growing polypeptide to SRP’s receptors in the cytosolic side of the endoplasmic reticulum. This incomplete polypeptide is then transported to the peptide translocation complex in the ER.
(c) Describe the structure of the ER Translocon and what are the associate proteins of the ER Translocon?
The ER translocon complex consists of large protein complexes such as Sec61, oligosaccharyl transferase complex (OST), the TRAP complex and TRAM ( membrane protein). The central region of the ER translocon houses the translocation channel Sec 61. The translocon functions as a channel through the hydrophobic membrane of the endoplasmic reticulum.
(d) Discuss the data of the paper in J.Cell Biol 1981 and Blobel’s results in the Cell, 1991 paper for the Translocon, and by Walter & Blobel in relationship to how the results led to the initially understood the role of the SRP.
The paper explores the translation induced inhibitory effect of SPR synthesis, reversal of the translational inhibitory effort displayed by microsomal membranes.
In order to understand this process, a translation system that was integrated with respect to polypeptide chain elongation, that was achieved by the addition of inhibitors of initiation right after the start of the incubation needs to used. This addition will allow the subsequent addition of membranes to be precisely timed with respect to chain length. (J.Cell Biol) Since protein synthesis stops after initiated chain are completed, it can be determined if the appearance of completed chains is due to chain completion of a previous arrested nascent chain and not of a new initiated chain. The 7-methylguanosine-5’-monophosphate was used in order to confirm the appearance of completed protein chains after the addition of the membranes. This is a result of completed chains that were previously elongated-arrested and not the newly initiated chains. Furthermore, when RM were added to the translation system after initiation, prolactin was formed only when membranes were added early, before the chain lost its ability for interacting with the membranes. When RM was added late, no prolactin was formed indicating there were no more nscent preprolactin molecules that were able to be translocated. Using SDS-PAGE analysis of the product of translation, it was determined that in the presence of SRP no preprolactin or prolactin was formed. Thus, prolactin mRMA translation can be arrested by SRP and the preprolactin synthesis will resume if we stop translation arrest and add K-RM. (J.Cell Biol). Because SRP was previously shown to recognize polysomes synthesizing secretory protein by information inside the nascent secretory protein. N-terminus segments are expressed outside the ribosome the size of a cleaved signal of preprolaction which means that the information required for the SRP to be able to recognize a polysome synthesizing secretory protein in inside the amino terminal signal. Additionally, SRP transmits information to arrest protein synthesis through ribosomal parts causing site specific elongation arrest. Other secretory proteins were found to be arrested by SRP and was released when microsomal vesicles were added and translocation occurred. Since SRP arrests the synthesis of secretory proteins, preventing its completion the the cytoplasm, only when membranes with translocation component sites are offered to the arrested polsome, it attaches to the membrane and synthesis then continues with translocation in the membrane. Preincubation of translocation competent microsomal membranes reduce translocation activity.
Blobel’s results in the Cell 1991 demonstrate the existence of a protein conducting channel in the ER membrane. Puromycin was used in the study for uncoupling a nascent polypeptide from it’s ribosome bound peptidyl tRNA by being incorporated into a carboxy-terminal nascent protein chain. (Cell 1991) These single channels were closed when the salt concentration was high because the ribosome detached from the membrane, showing that the attached ribosome keeps the channel open. The protein-conducting channel of the ER closes after each round of translocation, it follows that it opens for each new round. This paper describes protein-conducting channels in the ER that are revealed by puromycin and that close after detachment of ribosomes with high salt.
Compared to initial understanding, the 1981 and 1991 research papers helped with the discovery of signal peptides a few years later. Signal peptides form an integral part of protein targeting, a mechanism for cells to direct newly synthesized protein molecules to their proper location by means of specific location tag within the molecule. The two papers helped establish translation-inhibitory effect of SRP and the reversal effect of it by microsomal membranes by data and results discussed above. First paper shows how translational pathway is initiated when the signal peptide emerges from the ribosome and is recognized by the SRP. Then SRP stops further translation and directs the signal sequence-ribosome-mRNA complex to the SRP receptor, which is present on the ER. The second paper shows how by proving the existence of a protein-conducting channel in the endoplasmic reticulum membrane a mechanism for a complete cycle of opening and closing of the protein-conducting channel is suggested. Once membrane-targeting is completed, the signal sequence is inserted into the translocon. Ribosomes are then physically docked onto the cytoplasmic face of the translocon and protein synthesis resumes
Sources:
Simon, Sanford M., and Günter Blobel. “A Protein-Conducting Channel in the Endoplasmic Reticulum.” Cell, vol. 65, no. 3, 1991
Walter, Peter and Günter Blobel. “Translocation of Proteins across the Endoplasmic Reticulum III. Signal Recognition Protein (SRP) Causes Signal Sequence-Dependent and Site- Specific Arrest of Chain Elongation That Is Released by Microsomal Membranes.” The Journal of Cell Biology, vol. 91, no. 2, Jan. 1981
Alberts, Bruce, and John Wilson. Molecular Biology of the Cell. Garland Science, 2015.
3. Describe the Translocon system of the mitochondria.
The translocon system of the membrane is a protein complex located at the outer membrane of the mitrochondria . It functions by facilitating protein movement across the membrane of the mitochondria.
(a)How is translocation into the ER different from the mitochondria?
The translocation into the ER is different than that from the mitochondria in that the mitochondrial proteins are completely synthesized in the cytosol before being translocated by the post-translational mechanism. Another difference is the source of energy in translocation. The translocation into the ER across uses energy from the actual translation process – in which the growing polypeptide chian is feeded through the translocon channel. For translocation into the mitochondria membrane, the source of energy is from thebipartite Tom/Tim complex. In this Tim acts as the inner membrane translocon while Tom is the outer membrane translocon. For translocation into the mitrochondria membrane, there are three energy inputs needed. ATP hydrolysis by a cytosolic Hsc70 chaperone is needed first. It keeps the newly synthesized mitochondrial precursor protein unfolded in the cytosol. The second input is ATP hydrolysis by multiple ATP-driven matrix Hsc70 chaperones . These serve to drag the translocating protein into the matrix.
(b) Describe the proteins of the translocons and the accessory proteins involved in bring them into each compartment of the mitochondria.
Proteins of the translocons include TOM (translocase of the outer membrane) complex- this is found in the outer membrane and two TIM (translocase of the inner mitochondrial membrane ) complexes ( TIM23 and TIM22 complexes) – these are found within the inner membrane. Both the TOM and the Tim complexes consist of components which function as receptors for mitochondrial precursor proteins and other components that form the translocation channel. The TOM complex starts by transporting protein signal sequences into the intermembrane space as well as helping to insert transmembrane proteins to the outer membrane. The TIM23 complex then comes along and shuttles some of these proteins into the matrix space, while facilitating the insertion of transmembrane proteins into the inner membrane. The TIM22 complex is known to mediates the insertion of a subclass of inner membrane proteins such as Hsp70 the chaperone protein that transports molecules such as ADP, ATP, and phosphate. In addition to this, TIM22 of the inner membrane aid in the insertion of Metabolite carrier proteins.
β-Barrel proteins are chaperoned to the sorting and assembly machinery (SAM) and inserted to the outer membrane. TIM23, The presequence translocase, with the help of its associated motor (PAM) functions in the insertion of proteins to the inner membrane. The OXA complex, a protein translocator found in the inner mitochondrial membrane, mediates the insertion of inner membrane proteins synthesized within the mitochondria. The OXA complex also aids in the insertion of proteins that are initially transported into the matrix by the TOM and TIM complexes. Facilitated by the mitochondrial intermembrane space assembly machinery (MIA), intermembrane space (IMS) proteins are oxidized and folded.
(c) How is the mitochondrial signal sequence of the mitochondria different from that of the ER translocon system?
Within the ER translocon system, GTP hydrolysis during protein synthesis is known to drive the incomplete polypeptide from the membrane-attached ribosome across the ER membrane. GDP-GTP exchange as well as GTP hydrolysis fuel the insertion of incomplete secretory proteins into the translocon. The mitochondrial signal sequence acts to synthesize ATP using energy derived from electron transport and oxidative phosphorylation within the mitochondria. Mitochondrial Hsp70 also has a high affinity for the unfolded polypeptide chains. As such, Hsp 70 binds to an imported protein as soon as it appears from the translocator in the matrix. After this, the hsp70 releases the protein in an ATP-dependent step. The process of binding and and subsequent releasing is believe to provide fuel needed to complete protein import.
(d) . Explain Figure 1 in Mitochondrial contact sites as platforms for phospholipid exchange BBA 2017 in relation to mitochondria being formed from two distinct membranes, (MOM & MIM), what are the differences regarding there composition and function?
The communication between the ER and the mitochondria is facilitated by the mitochondria associated membranes. This is a linkage is important for regulation of lipid metabolism and transportation of molecules. The mitochondria produces phosphatidylethanolamine, phosphatidylglycerol, and cardiolipin and in this way plays a part in overall phospholipid biosynthesis. Due to the mitochondria’s role in phospholipid biosynthesis, it maintains highly active exchange of phospholipids with other cellular compartments. Figure 1 displays the transport of phospholipids between mitochondria and other organelles. It displays how the PE, PG and CL phospholipids are synthesized by mitochondria via modification of the head-group of precursor GPLs.
Mitochondria plays a crucial role in cellular lipid homeostasis due to the fact that they coordinate the synthesis important membrane phospholipids PE and PC.
The mitochondria acquire most of their lipids from the ER through the mitochondrial-associated ER membranes which act as membrane contact sites. The synthesis of PE and CL requires the transfer of PS and PA across the mitochondrial intermembrane space by enzymes present exclusively in the mitochondrial inner membrane. An abundance of PE and CL is required for multiple mitochondrial roles such as cristae development. The PE phospholipid is formed by the PS by the inner membrane which can then be exported to the ER for conversion to PC. Phospholipids can diffuse from one membrane to another as well as the fact that shuttling proteins can be used as form of lipid transportation between membranes. PA and PS from MOM and MIM of the mitochondria are transported by the Ups1-Mdm35 and Ups2–Mdm35. PA is converted to CL. This aids in the binding of Ups2 to the membrane and in delivering PS. PS is then converted to PE.
MOM (outer membrane) MIM (inner membrane). The MOM is the selectively permeable outer membrane of the mitochondria. It acts to transport molecules and acts as a partition between the organelles from rest of the cell. It also defines the intermembrane space between itself and the mitochondrial inner membrane. MIM is known to be a site of the electron transport chain as well as involved in aerobic respiration. The intermembrane space is the region between the inner membrane and outer membrane. In the intermembrane space, H+ ions accumulate to create a proton potential that facilitates the ATP energy formation. In addition to this, oxidative phosphorylation takes place within the inner membrane.
Alberts, Bruce, and John Wilson. Molecular Biology of the Cell. Garland Science, 2015.
Goldberg. Mitochondria, ER | Glycolysis-TCA Cycle | Urea Cycle 2018.pptx
Dimmer, Kai Stefan, and Doron Rapaport. “Mitochondrial Contact Sites as Platforms for Phospholipid Exchange.” Biochimica Et Biophysica Acta (BBA) – Molecular and Cell Biology of Lipids, vol. 1862, no. 1, 2017
5.
(a)Using the Receptor Mediate Endocytosis of LDL as a model, describe the vesicle formation, release of cargo (LDL) in the endosome and recycling of the receptor.
The LDL/lipoprotein receptors are produced within the golgi complex of cells. They then travel to the cell surface and gather into clathrin coated pits (clathrin on the inside). The receptor covered pit then joins the LDL along with this, multiple vesicles fuse to form endosome. LDL is released as the clathrin dissociates and there is a proton pumping action in the endosome membrane. This leads to a lowering of the pH which subsequently causes the LDL to dissociate from the receptor. The beta-propeller near the active, binding site of the LDL receptor causes this step. At neutral pH, it is linear and is able bind ligands. When the pH is lowered to an acidic pH, the beta-propeller site forms hairpins and prevents ligand binding. While folding into a closed conformation, the receptor remains in the vesicle form then returns to the cell membrane and repeats the process.
(b)How does the LDL bind to the LDL receptor
There are various domains on the LDL receptor. The core active site is on the N terminus of the receptor and functions to recognize and bind LDL to the receptor. The N terminal contains 7 sequential repeats which have very acidic residues. These aid in the formation of a coordination lattice with calcium. Repeated disulfide bonds forms crosslinked structures through the cysteine to keep the structural rigidity required for the constant and drastic pH changes. The beta propeller which is found next to the active site is responsible for the pH based acid/base conformational change. The seven sequential repeats of LDL receptors acts in recognition and in binding to LDL by the recognition of apoprotein 100B proteins LDL’s surface.
Brown MS, Herz J, Goldstein JL (August 1997). “LDL-receptor structure. Calcium cages, acid baths and recycling receptors”. Nature. 388 (6643): 629–30. doi:10.1038/41672Other source: Lecture Notes, Topic, Biosyn, Ketones, Cholesterol _ Regulation-2017.ppt (Goldberg, BCII lecture, 2017)
Rudenko G, Henry L, Henderson K, Ichtchenko K, Brown MS, Goldstein JL, Deisenhofer J (December 2002). “Structure of the LDL receptor extracellular domain at endosomal pH”. Science. 298 (5602): 2353–8. doi:10.1126/science.1078124
6. (a)Describe the ‘exocytosis’ of the GLUT 4 glucose transporter is controlled and signaling that placed it on the cell’s plasma membrane
GLUT4 can be described as an insulin-regulated glucose transporter which is responsible for insulin-regulated glucose uptake into fat and muscle cells. Insulin stimulates the transportation of glucose into the muscle and adipose tissue. The major glucose transporter expressed in these tissues is GLUT4. In the absence of insulin, the majority of GLUT4 is stored in small intracellular vesicles referred to as GLUT4 storage vesicles. After a meal, insulin is secreted by the pancreas and interacts with its receptor on the surface of adipocytes and myocytes which causes the activation of the canonical PI3K–AKT pathway.
Activation of PI3K–AKT pathway is required to elicit exocytosis of GLUT4 storage vesicles to the plasma membrane. This process involves microtubules and actin used to navigate GLUT4 storage vesicles in the direction of the plasma membrane, a tethering apparatus at the plasma membrane to simulate and capture GLUT4 storage vesicles at the cell surface as well as fusion machinery (SNARE proteins and SNARE-associated proteins) that fuses the GLUT4 storage vesicle lipid bilayer with that of the plasma membrane.
Stöckli, Jacqueline, Daniel J. Fazakerley, and David E. James. “GLUT4 Exocytosis.” Journal of Cell Science 124.24 (2011): 4147–4159. PMC. Web. 10 Mar. 2018.
(b) From the paper Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling Nature Comm 2014; what can you infer from the fact that actin inhibitor effect GLUT 4 expression on cell surface?
The inhibition on GLUT4 membrane insertion by Latrunalin B which prevents actin polymerization suggests that formation of new actin filaments is necessary for GLUT4 expression on the cell surface. Actin filaments are most likely involved in tethering of GSU’s to the plasma membrane.
(c) In Figure 1, a, b & c they identify Tmod3 as having been phosphorylate by Akt2. Explain the evidence of that confirmed this finding in 1a,b, & c and what is the significance of Tmod3 phosphorylation?
By tagged an active phosphorylated form of AKT2, a panel for its affinity proteins is shown. An immunoblot for ant-Tmod3 displays affinity of TMOD3 for AKT2 in its active, phosphorylating form. Additionally, a sequence analysis of TMOD3 revealed a single AKT consensus phosphorylation motif.
(d)What is the unique about the phosphorylation motif of Tmod3? (e)Inhibitors of PI3K or Akt had what affect?
The motif is unique to Tmod3. In that, there is no other tropomodulin isoform. Phosphorylation was ended when treated with P13K / AKT inhibitors
7.
(a) How does the NAD/NADH ratio play an important role in regulating glycolysis?
In glycolysis, a 10/1 The NAD+/NADH ratio is maintained. The limiting reagent for glycolysis is NAD and is required as an oxidizing agent for the pathway to continue. In this way, it determines whether or not glycolysis will occur. Essentially, the ratio of NAD/NADH is important to determine whether or not a pathway will be able to continue to completion. If there is more NAD than NADH, the reaction will proceed in a forward direction to form more NADH. This reaction is catalyzed by G3P dehydrogenase.
(b) How does the homolactic fermentation reaction in anaerobic mammalian cells help maintain the proper ratio of NAD/NADH?
The production of lactic acid uses the NADH formed during glycolysis and oxidizes it to NAD+ in an effort to reduce pyruvate into lactate. This action replenishes the concentration of NAD+ that is being used up in glycolysis. As such, the homolactic fermentation reaction aids in maintaining the high NAD/NADH ratio. In addition to this, DHAP is converted to glycerol-3-phosphate by glyercol-3-phosphate dehydrogenase which converts NADH to NAD+ to continue glycolysis. In aerobic cells, pyruvate is further oxidized to OAA hen reduced to malate producing NAD+ to also continue glycolysis.
(c)What are the pathways for maintaining this NAD/NADH in aerobic cells? In you answer remember to discuss thermodynamic and equilibrium.
In aerobic glycolysis, NADH enter the mitochondria because membrane is impermeable to NADH/NAD+. However, the cell is able to transfer electrons from NADH and restore NAD+ for glycolysis. Glycerol-3-phosphate dehydrogenase is converted from DHAP to G3P via the glycerol-3-phosphate shuttle. In this, Glycerol-3-phosphate dehydrogenase oxidizes one mole of NAD+ from NADH. Mitochondrial G3P dehydrogenase causes G3P to also be converted back to DHAP which leads to the reduction of FAD to FADH2. This step contributes two electrons to the ETC. Electrons from cytosolic NADH are also transported into the mitochondria via the malate-aspartate shuttle in liver and heart cells. Electrons from NADH are transferred to oxaloacetate to form malate which causes the production of NAD+ within the mitochondria. Malate is transported to the inner mitochondrial membrane and is reoxidized by NAD+ in the matrix to form NADH and oxaloacetate. NADH will be brought into the mitochondria by this shuttle if the NADH/NAD+ ratio is higher in the cytosol than in the mitochondrial matrix.
(d) How is the NAD/NADH ratio changed and why when a cell enters the reciprocal pathway to glycolysis, gluconeogensis?
The NAD+/NADH ratio changes when a liver cell enters gluconeogenesis from glycolysis due to the fact that glycolysis requires a NAD+/NADH ratio of 10:1 while gluconeogenesis requires a NAD+/NADH ratio of 1:10. Gluconeogenesis utilizes NADH and converts it to NAD+ to drive the reaction to form glucose while NAD+ is reduced to NADH in glycolysis. This is facilitated by the Cori Cycle in the liver cell where lactate that is created in muscle cells is transferred to the liver and then reconverted to pyruvate whilst converting NAD+ to NADH.
Goldberg, Biochemistry 2 (2017) Glycolysis-Gluconeogensis lecture
9. The keto acids of the TCA cycle are also the other substrates of the aminotransferase family. How does this family of enzymes play a role in maintaining the NAD re-oxidation of NADH to NAD.
(a) Use the amino acids alanine, aspartate and glutamate in your explanation.
Alanines participate in transaminase reactions with alpha-ketoglutarate to form pyruvate and glutamate. Both pyruvate and glutamate have the ability to influence the TCA cycle. Reductive amination, allows for α-ketoglutarate to be converted into glutamate. The amino group from glutamate can be transferred to other α-ketoacids by transamination reactions. As such, aspartate and alanine can be made from the addition of an amino group to oxaloacetate and pyruvate, respectively.
Glycolysis produces pyruvate. The pyruvate can then be converted to alanine and a-ketoglutarate through transamination with glutamate while fueled by the conversion of NAD+ to NADH. Nextly, alanine is shuttled to the liver and loses its nitrogen using alanine aminotransferase and glutamate dehydrogenase to form ammonia for urea synthesis and pyruvate.
The amino group of aspartate is transferred to a-ketoglutarate by aspartate aminotransferase to form OAA and glutamate. Glutamate is converted into a ketimine intermediate by glutamate dehydrogenase while utilizing NAD+ and water. Much of the energy generated by the oxidative steps of the cycle is transferred as energy-rich electrons to NAD+, forming NADH. Three molecules of NADH are produced for each acetyl group that enters the citric acid cycle.
In addition to this, electrons are transferred from NADH in the cytoplasm to OAA to form malate, which then travels across the inner mitochondrial membrane as well as malate may be reoxidized by NAD+ in the matrix to form NADH using malate dehydrogenase.
Goldberg, Special Topics, Mitochondria TCA cycle lecture
(b) What is the source of Ca2+ as a second message to increase the production of NADH in the TCA cycle?
Calcium from the endoplasmic reticulum is activated upon muscle contraction. The activation of the production of the ATP through the citric acid cycle is the signal that stimulates muscle contraction. Calcium ions regulate the citric acid cycle by activating pyruvate dehydrogenase, the first component of the pyruvate dehydrogenase complex reaction that forms acetyl CoA. Calcium ions also activate the enzymes, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase which catalyze the third and fourth steps of the cycle. When Ca+2 increases to activate the TCA cycle, it increases the level of pyruvate dehydrogenase.
(c) What activates the release of Ca2+?
Pyruvate dehydrogenase, isocitrate dehydrogenase, and a-ketoglutarate dehydrogenase in the TCA cycle activate the release of Ca+2.
(d) How is Ca2+ taken up into the mitochondria?
Calcium channeling occurs from the ER lumen to the mitochondrial matrix. The ER is the main intracellular Ca2+ store of cells, while the mitochondria shapes and decodes cellular Ca2+ signals by taking up and then releasing Ca2+ ions. Calcium release is mediated by the ryanodine receptors and the inositol trisphosphate receptors. Calcium exits the ER through the inositol trisphosphate receptor channel, enters mitochondria via VDAC, and then uses the mitochondrial Ca2+ uniporter to move into the mitochondrial matrix.
(e) Describe the structure of the mitochondrial OMM & IMM Ca2+ transporters. Why is the OMM Ca2+ transporter so unique?
Cytoplasmic calcium ions enter outer mitochondrial membrane via the voltage dependent anion-selective channel and are absorbed into inner mitochondrial membrane by mitochondrial calcium uniporter. Due to the fact that VDAC on the OMM is a selective ion channel regulated by the OMM electric potential, it is responsible for the rapid transfer of Ca2+ from the ER–mitochondria apposition, and their function results in high Ca2+ microdomains in the IMM. Chaperones and regulatory proteins (like Grp 75) control the formation of the ER–mitochondria junction. The capacity of VDAC transferring calcium ions is closely related to the number of VDAC on OMM. OMM controls permeability and thus it controls the release of apoptogenic factors, such as Cytochrome c. VDAC closure and OMM rupture serves as the cytochrome c release pathway that causes programmed cell death of apoptosis.
Goldberg. Special Topics, Mitochondria, ER, and Glycolysis-TCA Cycle lecture
10.
(a)What the functional significance of the Respirasome?
The respirasome is a supramolecular structure on the mitochondria composed of complex I, III, and IV. It transports protons across the mitochondrial membrane to create a proton gradient, which functions as the driving force for ATP synthesis. The functional significance of this is that it facilitates rapid transfer of the substrates in ETC. Adding to its functional significance, is that it also prevents harmful reaction intermediates from being released.
The flow of two electrons from NADH to FMN to Fe-S clusters to coenzyme Q through NADH-Q oxidoreductase (complex I) leads to the pumping of four hydrogen ions out of the matrix of the mitochondria. In accepting two electrons, Q takes up two protons from the matrix as it is reduced to QH2. This leaves the enzyme for the hydrophobic interior of the membrane. The NADH comes from both beta oxidation of fatty acids and tca cycle.
Q cytochrome c oxidoreductase (Complex III) catalyzes the transfer of electrons from QH2 to oxidized cytochrome c (water soluble protein) while pumping protons out of the mitochondrial matrix. The flow of a pair of electrons through this complex leads to the effective net transport of 2 H to the cytoplasmic side
Cytochrome c oxidase (Complex IV) acts to catalyze the transfer of electrons from the reduced form of cytochrome c to oxygen.
(b)Describe the structure of Complex 2 (succinate dh) and its relationships to the respirasome, glycolysis, pyrimidine synthesis and lipolysis.
Succinate dehydrogenase (Complex 2) generates FADH2, which feeds H+ & e- to Q cycle to generate H+ gradient at Complex 3. It forms a part of the IMM. Out of complexes I, II, III, IV, it is the only complex to participate in both TCA cycle and the ETC. It is comprised of four subunits, hydrophobic and hydrophilic (hydrophilic are in the matrix, while hydrophobic are in the IMM). The hydrophilic subunits are succinate dh (SDHA flavoprotein) and SDHB (iron-sulfur), and hydrophobic are SDHC and SDHD. Overall, complex II is a transmembrane-bound protein planted in the inner mitochondrial membrane where the ETC is. The main product of this reaction is FADH2 and Fumarate. The FADH2 produced from this reaction does not dissociate from the enzyme. Conversly, the FADH2 transfers the electrons to the iron-sulfur clusters and passes the electrons to Coenzyme Q (CoQ) that is part of the ETC which uses respirasome’s products by transporting electrons from several different complexes (I, II) to complex III. Coenzyme Q is also used in lipolysis and pyrimidine synthesis. In pyrimidine synthesis, CoQ is used in the step where dihydroorotate is oxidized by CoQ to orotate. The enzyme used is Dihydroorotate dehydrogenase. It’s the only enzyme in pyrimidine synthesis to be located inside the mitochondria rather than the cytosol. It transfers two hydrogen atoms to the enzyme because it is a flavoprotein similar to FAD. To reoxidize the enzyme, coenzyme Q takes them and utilizes them in the Electron transport chain. This ties ETC and pyrimidine synthesis together.
(c)Why does the oxidation of NADH+H+ generate 3 ATP and FADH2 generate only 2 ATP? Thermodynamics and Ox-Red are to be part of your answer.
c) The reduction potential of FADH2 is -0.22V, compared to the NADH with -0.32V. Less ATP is formed from the oxidation of FADH2 than from NADH since protons are not pumped from one side of the membrane to the other when it enters complex 2. There is also a larger energy difference between each complex with the use of the electrons from NADH (I, III, IV) than between each complex using FADH2 (II, III, IV). FADH2 to complex II doesn’t have an increase in redox potential.
(d) Describe the structure of the ATPasome and the accessory transporters. How does the ATP synthase utilize the proton motive force to generate ATP?
i) The ATPasome is another superstructural organization that, like the respirasome, is a collection of complexes. It includes the ATP synthase (can be called complex V), adenylate translocator, and phosphate carrier.ii) Protomotive force utilizes “Chemiosmotic theory” to generate ATP. Chemiosmosis is the movement of charged particles (electrons, H+ etc) across a selectively permeable membrane, which forms a gradient. It generates ATP by moving H+ ions across the membrane, against the electrochemical gradient. Acetyl CoA is oxidized in the matrix and couples to a carrier molecule such as NAD/FAD which is reduced to NADH/FADH2. These pushes the electron to the IMM, then to ETS. The energy in the electrons “pumps” protons from matrix to the IMM, storing electrochemical energy in the gradient. Then the protons move back across the ATP synthase, crossing the IMM, generating ATP.