BIO CBIO 3400 Fall 2012 Second Quiz Lechtreck Fechheimer QUESTION 1 25 POINTS DNA storage 2 pts production 2 pts a 10 Points List five cellular compartments or cell organelles Briefly describe its ultrastructural features and name its major function in keywords 1 Nucleus double membrane organelle Important for RNA transcription DNA replication and 2 Mitochondria double membrane organelle with inner membrane folded Important for energy 3 Endoplasmic reticulum single membrane organelle Important for synthesis of transmembrane and secreted proteins protein folding and protein N linked glycosylation 2 pts 4 Golgi apparatus single membrane organelle Important for protein modification O linked glycosylation M6P and sorting 2 pts 5 Lysosome Single membrane organelle Important for degradation 2 pts Or any other organelles such as plastid vacuole peroxisome proteasome ribosome etc b 5 Points How do we explain the origin of cell organelles such as mitochondria and plastids Endosymbiont hypothesis Mitochondria and plastids originate through the symbiosis of aerobic prokaryotic cells and host pre eukaryotic cells 2 pts Initially a prokaryotic cell is engulfed into a eukaryotic cell The host cell provides the outer membrane to surround the prokaryotic cells The prokaryotic cells were reduced to cell organelles such as mitochondria or plastids and lost their independence due to gene transfer in the host cell nucleus 3 pts c 5 Points List five facts supporting the theory hypothesis you described in the part b and 1 Have double membrane Inner one from the prokaryotic cell and outer one from the plasma explain why in keywords membrane of host cell 1 pt 2 Have circular DNA It s similar to prokaryotic DNA 1 pt 3 Lack histones Prokaryotic cells don t have histones 1 pt 4 Have smaller ribosome Similar to prokaryotic cells 1 pt 5 Start protein synthesis with formyl methionine Prokaryotic cells also use it to start the protein synthesis 1 pt Others such as polycistronic versus monocistronic mRNA mode of division involving Ftsz for plastids only mitochondria and plastids are phylogenetically related to prokaryotes etc d 5 Points Briefly explain the following terms in keywords nucleomorph a small reduced eukaryotic nucleus found in certain plastids 1 pt apicoplast a reduced non photosynthetic plastid found in most Apicomplexa Formed by secondary endosymbiosis It has four membranes 1 pt secondary endosymbiosis An eukaryotic cell engulfs another eukaryotic cell Eventually one is reduced to become an organelle of the host cell 1 pt stromatolites layered fossil structure formed by microorganisms such as cyanobacteria Provide the most ancient record of the life on Earth 1 pt compartmentalization rule a membrane separates a plasmatic and a non plasmatic compartment 1 pt NAME KEY Question 2 a 8 points Name four proteins or protein complexes required for co translational transport of a luminal ER protein into the ER and state the function of each 25 Points 1 sec 61 Translocon that protein moves through Also recognizes signal peptide 2 SRP Signal recognition particle Bind ribosome and nascent chain SRP 54 binds GTP Bind SR to take complex 1 to ER 3 SR SRP receptor Receptor on ER for the SRP Heterodimer of alpha and beta subunits Both subunits bind GTP 4 Signal Peptidase Cleave signal peptide from amino terminus of protein being transported Other answers also accepted b 9 POINTS 1 Name five proteins and their functions involved in protein folding in the ER 5 pts and 2 how cells respond when proteins fail to resume their native configuration 2 pts Bip HSP 70 Bind unfolded protein ATP hydrolysis promote folding Calnexin Membrane bound Bind protein with terminal glucose and prevent aggregation Calreticulin Soluble protein Bind protein with terminal glucose and prevent aggregation Protein disulfide isomerase Promote folding by catalyzing formation of proper SS bonds Proline cis trans isomerase Promote folding by catalyzing isomerization of prolines UPR Unfolded protein response Activate IRE1 kinase splice mRNA for transcription factor make more chaperones to promote correct folding ERAD ER Associated degradation Mark unfolded proteins with ubiquitin retrotranslocate out of ER to cytoplasm and degrade in proteasome UGGT could be included in part 1 or part 2 Other answers also accepted c 8 points Using a cell free translation system you study translocation and processing of prolactin To the cell free system you add mRNAs of variable length 50 150 codons due to the lack of a STOP codon the newly synthesized peptides will remain attached to the ribosome and indicate whether microsomes derived from rough ER were present in the translation assay 1 Explain the result of the depicted autoradiogram 5 points 2 The prolactin signal peptide is cleaved after 31 amino acids The channel in the ribosome occupied by the nascent polypeptide is 15 nm long the channel through the ER membrane is 5 nm long In an helix one amino acid extends for 0 15 nm whereas one residue occupies 0 35 nm when the protein has a fully extended confirmation Please calculate the conformational state of the nascent protein as it passes into the ER and explain your answer 3 points Increasing size of protein with more codons in mRNA No difference in size of protein microsomes from 50 110 codons Partial cleavage of signal peptide and complete cleavage of signal peptide with microsomes for 130 and 150 codons respectively 130 codons x 0 15 nm 19 5 nm Ribosome channel plus ER membrane is 15 5 nm 20 nm So nascent chain is alpha helix Should have seen cleavage by signal peptide with fewer codons if extended chain 0 35 nm residue Question 3 Key a Function transport from Golgi to ER 1 pt Golgi to Golgi 1 pt Coat composed of 7 subunits 1 pt Coatamer interacts with cargo receptors and transmembrane cargo 1 pt Coating regulated by ADP Ribosylation Factor ARF1 1 pt Arf1 is GTP binding protein GDP bound form is cytosolic 1 pt Membrane bound Arf GEF promotes GDP GTP exchange activating Arf1 leading to coat recruitment 1 pt Arf GTP is membrane associated N terminal myristoyl group inserts into membrane 1 pt Arf recruits effectors that change membrane lipid composition to promote curvature phospholipase D converts PC to phosphatidic acid 1 pt Uncoating is mediated by Arf GAP promotes hydrolysis of GTP 1 pt Arf GAP has amphipathic lipid packing sensor ALPS motif inserts into curved membranes 1 pt b 1 Golgi ER KDEL sequence recognized by KDEL receptor 2 pts Binds in lower pH