BIO 120 3rd Exam: Intracellular Protein Sorting and Maintenance of Cell Compartmentalization

BIO 120 3rd Exam: Intracellular Protein Sorting and Maintenance of Cell Compartmentalization Compartmentalization - Due to network inside the cell Eukaryotic Cell Compartment Assembly of organelle depends on protein transported to it Highly regulated, specific and targeted Intracellular Compartment Nucleus continuous in cytosol (topologically continuous) o Molecules can move from one place to another without crossing the membrance Secretory and endocytic network o Vesicles, endosomes, RER, golgi apparatus Mitochondria, plastids, and peroxisomes Nucleus continuous to ER continuous to Golgi apparatus Protein Transport Mechanisms GATED TRANSPORT Use of the nuclear pore complex (not membrane) TRANSPORT ACROSS THE MEMBRANE Use of trans membrane protein translocators Chloroplast, mitochondria, peroxisomes VESICULAR TRANSPORT Series of budding and fusion ER, GA, Endosomes and lysosomes Directionality of Protein Transport All proteins are started to be synthesized in cytosol Cystosol mitochondria Plastids Peroxisomes ER nucleus Golgi Apparatus (complete synthesis to ER) Endosomes Lysosome cell membrane General Requirements for Protein Transport Signal sequence Receptor Translocator Energy source 1. Signal Sequence Sequence of a.a. in protein itself which target the protein to its compartment Aka signal peptide, targeting sequence 2 possibe locations: o 1. Terminally located – amino (N) or Carboxyl (C) terminus ▪ Cleaved by SIGNAL PEPTIDASE when transported ▪ Not essential for protein o 2. Internally located ▪ Found in many segments ▪ Not cleaved ▪ They form folding 2 or 3 conformation that direct to target specific organelle ▪ They are recognized by receptors 2. Receptors Located either on cytosol or organellar membrance Show molecular complementation with the signal sequence Guides the protein to their correct compartment Show specificity on class of proteins Go back to original location (reuse/recycle) 2 types of protein transport in terms of timing with respect to translation: a. Post Translational import Protein synthesis by free ribosomes in the cytosol Transported/imported to respective compartment/organelle b. Co-translational import Protein synthesis by ribosomes docked in the ER membrane o Because of ribosomes attach to it that perform translation o During translation, these proteins are being at the same time transported to the ER lumen PROTEIN TRANSPORT INTO THE NUCLEUS Translation proteins for replication and transcription CYTOPLASM NUCLEUS Large one; have nuclear envelope that have outer membrane which is continuous to rER membrane Both membranes have ribosomes attach to it INNER NUCLEAR MEMBRANE o Lined with nuclear lamina NUCLEAR LAMINA o Network or fiaments where chromatin are attached NUCLEAR PORE COMPLEX (NPC) o Along the nuclear envelope NUCLEAR LOCALIZATION SIGNAL (NLS) o Mixed of Pro-Lys-Arg o Note: Memorize single letter of aa! Transcription yields to 3 RNAs exported out from the nucleus into the cytosol because translation takes place in the cytosol There are proteins initially imported to the nucleus, exported back out into the cytosol Some proteins (ribosomal proteins) are synthesized in the cytosol, imported to the nucleus then exported back to the cytosol Experiment: Cytosol is fluorescing bec. It is where fluorescence is observe Know principle about immunolabeling, chemiluminiscence NUCLEAR PORE COMPLEX (NPC) Not just a pore Made up of 100 types of polypeptide 2 parallel rings, 8 subunits o ANCHOR PROTEIN ▪ Attach the whole complex into the nuclear envelope o TRANSPORTER ▪ Actual shuttle of transport/assist transport at the middle part of the spoke o SPOKE ▪ Connect together all the components of the complex MAMMALIAN CELL NUCLEUS ● Have thousands of NPCs ● Nuclear envelope not smooth; bec. Of NPCs Responsible to both passive and active transport (small molecules including DNA and RNA nucleotides, histones (for chromosome packaging)) ENZYMES o Actively transported, requires energy and they contain signal sequence which is generally called NUCLEAR LOCALIZATION SIGNAL (NLS) o Amino acid that makes NLS: ▪ Proline (P), Lysine (K), Arginine (R) How specific signal sequence are? ● Signal sequences cannot be imported to compartments remain in the cytosol if they contain other type of AA ● NLS can be located anywhere in the protein terminally IMPORTIN Receptor in the cytosol Once importin encounter protein with NLS, it recognizes and binds with NLS which is molecularly complementary Thus, 2 will now be targeted and diffused as mediated by NPC Importin-NLS complex recognized by ran GTP (found inside the nucleus) Ran of Ran GTP o Recognize and binds with the importin; changes conformation o Thus, it cannot bind to NLS protein; the NLS containing protein is released in the nucleus IMPORTIN - RAN-GTP COMPLEX Targeted again in the NPC and exported out in the cytosol Once in the cytosol, ran hydrolyzes GTP to become GDP o Ran changes conformation, therefore cannot bind to importin, releasing the importin o Importin return to original shape where it can bind to another protein REMEMBER: Ran GTP inside the nucleus; Ran GDP in cytosol Bind to receptor for transport and provide directionality in transport of proteins NUCLEAR EXPORT SIGNALS (NES) Signal sequences to direct proteins out of the nucleus (in the cytosol) Rich in leucine that serve as the signal that it should be exported out of the nucleus This protein contain NES recognized by exportins The exportin in free form canot bind to NES yet Ran GTP bind to free exportin, exportin changes conformation to a form complementary to NES so that they can now bind together Molecule that have Ran GTP, exportin and protein to be exported Once in the cytosol, ran hydrolyzes GTP to GDP then changes conformation Releasing the exportin bec it is not longer complementary it also releases NES protein now in the cytosol Ran GDP recycled back in the nucleus to become Ran GTP SHUTTLING PROTEINS protein goes in and out of the nucleus contains both NLS and NES depends on the rate where the protein will be found mostly o import export = inside the nucleus o import export = found in the cytosol Some proteins continuously goes in and out of the nucleus; some proteins are regulated. HOW? 1. Turning on and off the specific signal sequence (NLS or NES) a. If protein is to be imported, NLS should be turned on, NES is off. HOW? i. Phosphorylation of aa closer to signal sequence (NLS/NES) 2. Association of such protein with inhibitory cytosolic proteins a. Inhibitory proteins attach this protein to a substrate preventing the movement even with NLS/NES b. Suppress NLS i. Attach NLS to cytoskeleton. Thus, it cannot move 3. This inhibitory protein will mask the NLS so that it wouldn’t recognize by importin; so they can stay in cytosol a. A certain stimulus is needed to release the inhibitory with NLS; there they would be imported Exported of tRNA, snRNA, microRNA – exported from nucleus unto the cytosol mRNA out of the cytosol is different in mechanism mRNA are also assoc. to larger protein called mRNA ribonucleoprotein complexes being exported as part of mRNA (mRNP) hundreds of different types of proteins MRNP will be now be targeted into NPC o Fibers - strips away the protein as this mRNP will move and exported out of the cytosol o Fibers will trim the nuclear protein that are assoc. with the mRNAs o Because of stripping, some nuclear protein will now be in the cytosol o Nuclear proteins contains NLS, recognizesd by importin then it will go back to the nucleus o Its active thus requiring energy provided by ATP hydrolysis The import of nuclear protein trimmed off by mRNP then imported to nucleus, ran GTP is indirectly involved. HOW? ● mRNA synthesized in cytosol and imported in nucleus thru importin and released by Ran GTP ASSEMBLY OF RIBOSOMAL SUBUNITS rDNA gene, 1 located (large) in the nucleolus, 1 in the cytoplasm = 2 rDNA transcribed to form precursor rRNA in the nucleolus in the cytosol, ribosomal proteins are synthesized part of small/large subunits o and post translationally imported into the nucleus making use of receptors, ran GTP, they contain NLS now interact with precursor rRNA complex of rRNA and proteins will undergo cleavage and modifications some rRNA and proteins are lost (no longer part of the final product) in the nucleoplasm , the smaller unit transcribed to form 5S rRNA rRNA and proteins in the nucleolus will now move out in the nucleoplasm large subunit will now bind to 5S, remaining 1 will be exported out using the nucleus again using NPS to be recognized by exportin everything will be in the cytosol (small and large subunits) small and large subunit will form ribosome in translation process it initiates, other use 2 sep. subunits there are ribosomes that are cytosolic (free in cytosol or assoc. with ER membrane/ be part of the ER) other ribosomes in the organelle (mito) have own set of ribosomes as well as chloroplast these organelles will have heterogeneous ribosome with several types of ribosomes in prokaryotic ribosomes CYTOSOLIC PROTEINS no signal sequence, not directed to any organelles, stay in cytosol in cytosol they will undergo modifications or covalent modifications: o GLYCOSYLATION- gain CHO moiety o Gain enzyme (non protein but organic; help activate a particular protein; permanent) o PHOSPHORYLATION - Gain phosphate group (or dephosphorylation) o METHYLATION – gain methyl group o ACETYLATION – gain acetyl group Cytosolic proteins may go to plasma membrane if there is where they will be functional DEGRONS Signal protein for degradation of protein Composed of destabilizing aa Found in the amino terminus of protein to be degraded Degrons are recognized by UBIQUITINE UBIQUITINATION – protein digestion/degradation STEPS IN UBIQUITINATION 3 Enzymes: E1, E2, E3 Enzyme 1 (E1) o Ubiquitin Activating Enzyme o Attach itself to unit of ubiquitin with required ATP o Ubiquitin o Complex made up of ubiquitin plus enzyme Enzyme 2(E2) o Ubiquitin Conjugating Enzyme o Ubiquitin transferred from E1 to E2 o Facilitate transfer of Ubiquitin from E1 to itself o Product: Ubiquitin plus E2 complex Enzyme 3(E3) o Ubiquitin ligase o Ubiquitin ligase attach to a substrate (protein with degrons) o Catalyse transfer if ubiquitin from E2 to substrate o Ubiquitn binds to exposed lysine of proteins with degrons o Polyubiquitination ▪ Additional of ubiquitins will be attached serve as signal to 26Sprotease to degrade 26S Proteosome have 2 components: 19s regulatory/ATPase complex 20s core o 4 rings (with 7 Subunits) o α rings – first 2 rings o β rings – responsible for actual digestion 19s – initially bind the protein to polyubiquitin TAG, unfold protein and protruded to into β rings Degraded because they are damaged proteins (no longer needed) IMPORT AND EXPORT OF PROTEINS TO MITOCHONDRIA Signal sequence in amino terminus (made up of AA that are BASIC, HYDROXYLATED AND HYDROPHOBIC) ● Basic because the translocation machinery imported to the mito have acidic domain; they are electrostatically attracted Specifically aa S, R, K, L (Serine, Arginine, Lysine, Leucine) This signal seq are cleaved by signal peptidase in mitochondria Internally located ss (not cleaved, not yet characterized) Proteins meant in diff. sub compartments of mito will have diff combination of signal sequences which provides specificity TRANSLATION MACHINERY FOR IMPORT TO MITOCHONDRIA TOM (Translocate Machinery in OM) o Receptors that can recognize transport found in the OM o 2 types: ▪ TOM 20- receptor for NMTS or with internally located protein ▪ TOM 70 – receptor for internally located protein o 3 possible destinations: ▪ Laterally inserted in OM ▪ Pass on on SAM complex then insertin it to --- ▪ Compartment beyond OM (IM, IS, Matrix) TIM (Translocate Machinery in IM) o 2types: ▪ TIM 22 – specific for subclass of protein to be inserted in the inner mito membrane ▪ TIM 23 – recognize all protein with NMTS ▪ Matrix proteins ▪ Some are inserted to IM/IS OXA o Proteins found in outer membrane o Inserts protein in inner membrane o Recognize in matrix; insert matrix protein to inner memebrane o Recognize protein synthesize in matrix to be inserted in IM CHAPERONES 2 sets: o CYTOSOLIC HSP 70 ▪ Heat shock ▪ Bind to proteins after synthesized by ribosomes so that it is not folded yet ▪ Prevent aggregation o MITOCHONDRIAL HSP 70 ▪ Found in mito matrix ▪ Bind to polypepide then pulled to matrix ▪ Some proteins not exposed to HSP 70 if not bound in matrix ▪ Have specific signal seq STEP BY STEP: PROTEINS INTO MATRIX N-MTS o N-terminus (signal sequence) o Recognized by receptor TOM 20 HSP 70 associate itself; preventing folding or associate itself to other peptide/recognized by receptor in outer mito membrane (TOM 20) Transferred tor pore of TOM complex HSP 70 must release the polypeptide (required ATP hydrolysis) Another set of HSP 70 will now attach to polypeptide Pull polypeptide to matrix They will still detach which requires ATP again Exposed to matrix either partial or full Once in the matrix, polypeptide may now fold into functional form with an aid of another chaperone molecule (HSP 60) o HSP 60 – aid in folding PROTEINS TO OUTER MEMBRANE No NMTS Have internal sequence – TOM 70 2 routes: depends on secondary conformation of polypeptide o α helical domain in polypeptide ▪ After recognition with TOM 70, it will be released to pore-TOM complex ▪ TOM complex released it laterally to outer mito membrane to be inserted in outer membrane ▪ Should have trans membrane domain o Β barrel domains ▪ TOM 70 TOM Complex SAM Complex o SAM complex ▪ Aid in insertion of OMM PROTEIN IN INTERMEMBRANE SPACE 2 Pathways: depends on SS N MTS also internal SS o Function as stop transfer sequence (moment comes contact with pore, transport of its own stops) it has a site for cleavage Transfer stops TOM 20 TOM Complex TIM 23 Complex Internal sequence may interact with TIM 23 contact with IM space targeting sequence Arrest/stop transfer It will be laterally released into inner membrane Stops transfer sequence aa o Hydrophobic aa enzyme that cleaves protein No NMTS, have internally located SS o Stop transfer o Receptor is TOM 70 TOM complex release in intermembrane space PROTEIN IN INNER MEMBRANE 3 pathways: o 1st: N MTS, internally located SS (stop transfer sequence – no cleavage site ▪ Same route as with 1st route of intermembrane space ▪ Laterally release to inner membrance, no cleavage ▪ Stop sequence will be trans membrane domain o 2nd: N MTS, and internally located SS ▪ No stop transfer sequence ▪ OXA targeting sequence ▪ Recognized by OXA ▪ OXA ● Translocon complex in inner mito membrane ▪ Terminally located SS ▪ TOM 20 TOM complex TIM 23 complex HSP 70 (pulling into matrix) whole polypeptide into matrix cleaved OXA complex mediate insertion into inner mito (insertion twice) o 3rd: no NMTS and several internally located SS ▪ TOM 70 TOM complex to pass outer MM TIM 22 complex Aid in insertion into inner (multipass inner membrane protein) membrane ENERGY Secure for transport 1st: spontaneous Import / pathway o Does not require translocation complexes require energy; no ATP hydrolysis ▪ Not conclusive; may still require ATP/ membrane potential 2nd o Require translocation ▪ Complexes to inner and outer mito mem o Requires HSP 70 in cytosol and mito o Requiring ATP o Dependent on electrochemical gradient H+ ions o PMF electrons from ETC in inner mito ▪ P side – facing the intermembrane space ▪ N side – facing matrix o PMF as source of energy for transport of protein into mito o Use ATP and PMF 3rd: General Pathway o Require translocation complexes o Requires HSP 70 (cytosolic and mito) o ATP require (cytosol and matrix) o Another factor required o Depends on ATP and PMF o Require membrane potential CHLOROPLAST Same mechanism with mito Post translational import Receptor in outer chloroplast TOC and TIC – not homologous ; receptor analogues; completely to each other o Same function with TIM and TOM o Different in structure Use chaperones so polypeptide will not fold E source: PMF and ATP/GTP Outer and inner membrane, intermembrane space, stroma (corresponds to matrix) Similar pathways Have addtl compartments: o Thylakoid lumen and membrane PMF: found in thylakoid membrane bec. ETC can be found there P side: stroma/ thylakoid lumen N side: How proteins be targeted to this compartment? Protein should have signal sequence in amino terminus to be targeted in the stroma Also should have signal seq targeted to the thylakoid Receptor TOC complex outer TIC complex inner May require ATP Also have signal peptidase in the stroma Transloacation to inner and outer has GTP/ATP hydrolysis, not by PMF bec. There is no PMF in inner chloroplast membrane TRANSPORT OF PROTEIN INTO THE THYLAKOID 2 Pathways: o SEC Pathway ▪ Requires homologs of SEC proteins ▪ SEC proteins – found in bacteria membrane ▪ Function is to mediate protein transport ▪ Found in thylakoid membrane ▪ E source: ATP and PMF in thylakoid membrane o SRP like Pathway ▪ Signal Recognition Particle ▪ Protein present in the stroma ▪ Protein in homologues of SRP ▪ E source: ATP and PMF Twin Arginine Translocation Pathway (TAT) Two Arginine residues in signal sequence are very important in order for protein to follow this pathway Twin Arginine – 2 arginine residues in signal sequence directing to protein to the thylakoid E source: PMF Spontaneous Import into the Thylakoid Receptor in stroma Translocon in membrane No known E requirement