MCB Study Notes For the Final.docx

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Molecular and Cellular Biology
MCB 2050
Georgevander Merwe

MCB Study Notes- FOR FINAL The Nucleus  eukaryotes have a membrane bound nucleus with a nuclear envelope, nuclear pores and an internal nuclear structure  prokaryotes have a region where the chromosome is located (nucleoid) Characteristics  irregular shape, one per cell  largest organelle Functions 1. Compartmentalization of cellular activity and genome 2. Coordination of cellular activities  Every process that occurs from the body comes from nucleus Structure  Nuclear envelope- nuclear membrane, lamina and pores  Nuclear content-chromatin, nucleoplasm, nuclear matrix, nucleolus Nuclear envelope (NE): Structure  2 phospholipid bilayers – have intermembrane space, outer membrane binds ribosomes and is continuous with the RER  inner membrane has unique proteins, integral membrane proteins that connect nuclear lamina  intermembrane space is continuous with ER lumen  inner and out membranes join at pores Nuclear Envelope: Function  separates nuclear content from cytoplasm- genome from cytosol, transcription from translation  Selective barrier (size specific)- establishes composition of nucleus  Binds nuclear lamina-structural framework from nucleus Nuclear Lamina Structure  Thin meshwork of long filament like proteins  Bound to inner surface of NE Functions  Support structure for NE  Scaffold for chromatin and nuclear matrix attachment  Mutations in lamin genes responsible for several human diseases (Hutchinson-Gilford progeria syndrome- premature aging in children due to point mutation in lamin A gene and destabilization of lamina) Nuclear Pores  Inner and outer membranes of nuclear envelope fuse into pores  Gateways between cytoplasm and nucleoplasm Nuclear Pore Complex (NPC) BIG!!!!!!  Complex supramolecular complex, fits into the pore, and reduces functional diameter  Extends into nucleoplasm and cytoplasm Structure  Highly conserved- integral and peripheral outer and inner membrane proteins  Octagonal symmetry around large aqueous central channel  Central scaffold- anchors complex to nuclear envelope  Inner surface of channel is lined with filament like .. FG nucleoporins (NUPs) and a lot of a.a’s (phenylalanine-glycine residue repeats) 1  FG repeats have disordered secondary structure- extendable and flexible, extend into central canal, hydrophobic mesh (limits size of things passing)  Cytoplasmic and nuclear rings  Cytoplasmic filaments- involved in nuclear receptor cargo protein recognition and import  Nuclear basket- on nuclear side, involved in nuclear receptor cargo protein import and export Size Exclusion at NPC  Small molecule move freely through by passive diffusion so they move quickly through the NPC  Larger molecules greater than 40 kDa need active transport so they move much slower through (RNAs, proteins etc.) Nuclear Import of Nucleoplasm  Nucleoplasm=nuclear protein  Nucleoplasm is made in cytoplasm, associates with cytoplasmic filaments and goes to the nucleus Nucleocytoplasmic Transport via NPC  Cytosol-to-nuclear transport- need energy, protein receptors, targeting signals  Most nuclear imported proteins have nuclear localizing signal (NLS)  NLS- specific sequence of a.a’s recognized by nuclear receptor proteins, several diff types of these based on a.a. sequence  Cargo molecule- destined for the nucleus but made in cytoplasm NLSs  Classic NLS- one short +vely charged a.a residues  Bipartite NLS- two short +vely charged a.a’s, and a spacer sequence (only basic residues at the end)  What is NLS? A.a sequence that is needed for cytosol to nuclear transport  If sequence is mutated the protein does not go to the nucleus (NLS necessary)  if the sequence linked to a non nuclear protein is capable of redirecting the resulting fusion protein to the nucleus (NLS is sufficient)  NLS experiment- in ARC 1- is a protein needed for plant pollination and shuttles between cytosol and nucleus but needs a classic NLS and NES (export signal) Other Required proteins for Cytosol-to-nuclear transport 1. Transport receptors (Karyoferins)- move protein cargo across nuclear envelope. Into the nucleus called importins, out of nucleus called exportins 2. Small G-binding proteins (G-proteins)- Ran is a Ras related nuclear protein, Ras stands for rat sarcoma proteins. G-proteins are molecular switches in the transport process Conformational Changes in G Proteins  G proteins have weak intrinsic GTPase activity- GTP binding and hydrolysis cause conformational changes  GTP hydrolysis (loss of phosphate) causes inactivation with the help of GAP-GTPase activating proteins (in cytoplasm) which enhance catalytic activity  GTP binds (1 phosphate NOT added) and reactivates it with the help of GEF- guanine exchange factors (in nucleus) G Proteins  Ran is a GTP binding protein- cant stay GTP long. Has two states- active and inactive  High Ran-GTP in nucleus and low amount in cytoplasm, gradient maintained by accessory proteins  Cytoplasm-GAP hydrolyzes GTP to GDP to provide energy for transport and Ran GDP translocates to nucleus  Nucleus- GDP in Ran is exchanged for GTP by GEF Nuclear Transport- multi step process 1.-importin in the cytosol recognizes nascent NLS containing cargo proteins -importin= heterodimeric protein because it has two subunits, beta and alpha 2 - importin alpha subunit binds and recognizes basic residues of NLS protein 2.-the cargo protein importin receptor complex moves through cytosol to nucleus (importin can bind to cytoskeleton) -at surface of nucleus importin beta subunit binds to a cytoplasmic filament in the NPC 3.- complex is translocated through NPC channel by interacting with FG domains which dissolve the FG network and stretch for passage of cargo 4.- complex associates with nuclear basket and binds to Ran-GTP, changes protein folding, (via importin beta) and is released and disassembled from the NPC 5.-Ran-GTP bound importin beta subunit goes back to cytosol because more Ran GTP is in the nucleus. In the cytosol the GTP is hydrolyzed and Ran-GDP is released from importin beta. Ran-GDP moves back to nucleus because concentration gradient and converted back to Ran-GTP by accessory protein What is the Fate of Importin alpha in nucleus?  Importin alpha binds to exportin and so does other cargo protiens by nuclear exporting signals. Most commone NES is leucine based motif.  importin alpha exportin complex binds Ran-GTP because its high in nucleus, Ran-GTP and this makes the complex assembly stable. This complex now goes to the cytosol because of the concentration gradient  Ran GTP is than hyrdrolyzed in the cytosol to Ran-GDP by accessory protein and is released from exportin which than causes release of importin alpha  importin alpha reused for import, Ran GDP goes back to nucleus and is converted to Ran GTP  exportin moves back to nucleus to be reused ***some proteins are imported without NLS because they piggyback nuclear protein import by binding to NLS containing protein in the cytosol Nuclear Content Nucleoplasm  has ions, cofactors, and nucleotides  has more than 30 specialized regions with specific functions  has nuclear matrix Chromosomes  chromosomes in interphase are in certain subdomains in nucleus  location of gene is related to its activity- most actively transcribed genes are at periphery of chromosomal subdomain  interchromosomal channels- regions between domains that are barriers to prevent unwanted DNA-DNA and or DNA-protein binding interactions  active genes (chromatin) from different subdomains extend into interchromosomal channels to form transcription factories where transcription factors are concentrated and nuclear speckles Nuclear Speckles  subdomains where mRNA splicing factors are concentrated  usually in interchromosomal channels next to or overlapping TFs  they are numerous and highly dynamic- they grow in size and # dramatically Nucleolus  irregular shaped, dense and granular appearance (not membrane bound)  size and number depend on metabolic activity of cell  Function- ribosome biogenesis  Site of ribosomal DNA gene transcription and rRNA processing (large-60 and small-40 subunit) 3  Initial stages of ribosomal subunit assembly- final assembly takes place in cytosol  Most expensive process in the cell is protein synthesis Nuclear Matrix  Insoluble fibrillar like protein network through out the nucleoplasm  Analogous to cytoskeletal network in the cytosol (microtubules and microfilaments)  Structural role and serves as a scaffold for organizing nuclear subdomains and anchoring protein factors Cell Cycle Two main phases: 1. Interphase-has three stages  G1-Gap1, cell performs normal cellular activities and responds to environment  S-Synthesis, DNA replication and increased synthesis of required factors for chromosome duplication  G2-Gap2, cell grows and prepares for M phase  G 0Gap 0, non dividing cells (most of cells in body) arrest in G1 and do not go to S phase 2. M Phase- mitosis  Prophase, metaphase, anaphase, telophase, and cytokinesis (mother cell divides into two daughter cells)  Prophase includes- chromosome duplication, mitotic spindle formation, reversible breakdown of nuclear envelope Control of the Cell Cycle  Cancer=inability for cell to regulate its own division  Checkpoints (regulation at distinct stages)- ensures cell cycle is progressing properly  Checkpoints- (1) end of G1 to ensure it can go on to DNA replication (2) end of G2 to ensure it can go onto M phase (3) End of Metaphase cell commits to chromosomal segregation- makes sure there is not unequal # of chromosomes in daughter cells  What is responsible for stimulating transitions from one phase to the next? Cytosolic mobile proteins –CDKs and Cyclins  CDKs- kinase enzymes-phosphorylate various target proteins in order to turn them on or off  Cyclins- concentration varies in a cyclical fashion during cell cycle, bind to CDKs and change CDK formation thereby regulating its activity  During early interphase (G1)- cyclin is low and so is CDK activity  During end of G2- cyclin high and so is CKD activity and there is phosphorylation of target tissues CDK target nuclear proteins at end of G2 and early metaphase:  Histones and condensins- phosphorylation leads to chromatin packing and chromosome condensation  Lamins- phosphorylation leads to disassembly of nuclear lamina (no more support structure)  NUPs- phosphorylation leads to disassembly of NPC Nucleus during mitosis- Open Mitosis  Higher eukaryotes  Nucleus is completely disassembled by metaphase  Two daughter nuclei reassembles during telophase  Cyclin low, CDK activity low, dephosphorylation of NUPs and lamins  Reforming nuclear lamina, envelope and NPC, reimport of soluble NLS proteins from cytosol Closed Mitosis  Lower eukaryotes  Nuclear envelope remains intact during mitosis, still have CDKs but don’t have CDKs to take apart nuclear envelope Control of Cell Cycle  Diff cyclins bind to same CDK  Oscillations in different [cyclin] during cell cycle leads to changes in CDK activity 4  Oscillations of [cyclin] due to relative rates of protein synthesis and degradation at diff points in cell cycle (end of phase cyclin is high, start of phase cyclin is low)  The decrease in cyclin after the start of M and S phase is because of a decreased synthesis of new cyclin proteins and degradation of pre existing cyclin proteins  CDKs are inactivated by phosphorylation  Pre existing cyclins also prevented from targeting to the nucleus and cannot activate CDKs in nucleus Nucleoplasmic Transport of Cyclins  Cyclins shuttle between cytosol and nucleus so they contain NLS and NES  Up to and during G2 NES>NLS  End of G2 NLS>NES because cell is actually needed here and you want cyclin in M phase Main Mechanisms of CDK Regulation (5) Cyclin Binding  Cyclin induces change in cdk conformation- activates cdk Proteolysis of Cyclins  Ubiquitin ligase adds ubiquitin to cyclins  Ubiquitin cyclins degraded by proteasomes-irreversible Cdk Phosphorylation State  Phosphorylation of cdk-mitotic cyclin complex inhibits cdk  Dephosphorylation of cdk-mitotic cyclin complex activates cdk  Cdk Inhibitors  Proteins that bind and block cdk-cyclin complex-degradation of inhibitor activates cdk Subcellular Localization of Cyclins  Mitotic cyclin B1 needs to be nuclear for cells to initiate mitosis Endomembrane System Organelles in endomembrane system differ: 1. certain proteins 2. unique activities 3. compartmentalization and functional diversity- do same thing in certain locations 4. conserved in eukaryotes 5. dynamic structures – change constantly, ER may not always be near nucleus Structures: 1. ER 2. Golgi 3. Endosomes 4. Lysosomes/vacuoles 5. Secretory granules 6. PM Vesicle Transport Step 1  Cargo containing vesicle buds off the donor membrane compartment  Vesicle cat proteins select which donor membrane and lumenal cargo proteins can enter Step 2  Nascent vesicle transported through the cytosol to the recipient membrane compartment  Vesicle receptor proteins regulate intracellular trafficking to proper recipient membrane  Involves molecular motors and cytoskeleton highways Step 3  Vesicle fuses with the proper recipient membrane compartment  Receptor proteins also regulate vesicle recipient membrane fusion  Vesicle membrane and cargo proteins go into recipient compartment  Bulk flow=components of vesicle that should not be there- reverse mechanism sends them back with the help of other receptor protein 5 Step 4  Process repeated, and can occur in reverse direction Distinct Trafficking Pathways Biosynthetic Pathway  Materials transported from ER to golgi, to endosomes, and than to either lysosomes/vacuoles, or to the PM Secretory Pathway (2 types) 1. Constitutive Secretion- ER derived materials are transported from golgi to PM and/or released out of cell. Secretory transport vesicle membrane components are incorporated into PM and vesicle luminal cargo is released into extracellular space 2. Regulatory Secretion- occurs only in specialized cells. ER derived materials from golgi are stored in secretory granules. When signal present granules fuse with PM and release cargo out of cell ex-neurotransmitters into synaptic cleft Endocytic Pathway  Material move into cell  Materials from PM (receptor proteins for degredation) and/or extracellular space are incorporated into cell and than transported to endosomes and to lysosomes –material either recycled or broken down in lysosome Endoplasmic Reticulum (ER)  Complex network of membrane enclosed rod like tubules and sheet like cisternae , organelle with largest SA Structure  Lumen-aqueous space inside ER tubules and cisternae  Tubules and cisternae shapes are regulate by reticulons (ER integral membrane proteins that have hair pin secondary structure and regulate ER membrane curvature)  Is a highly dynamic network- tubules and cisternae are in constant flux (bending, fusion, fission)  ER contains multiple subdomains (unique functions) Subdomains: 1. Rough ER- cisternae with bound ribosomes, involved with protein and membrane phospholipid synthesis 2. Smooth ER- curved tubules lacking ribosomes, involved in Ca 2+storage and hormone synthesis 3. Outer nuclear membrane- continuous with RER, has NUPs and attached ribosomes 4. Mitochondria and PM associated membranes- ER makes direct contact with them, involved with membrane lipid exchange 5. ER Exits sites (ERES)- transport vesicles bud off from the ER en route to golgi Protein Synthesis (two main sites for it) 1. Free ribosomes in the cytosol- nascent protein may stay in the cytosol, or may go to its proper intracellular destination 2. ER membrane bound ribosomes- soluble or membrane protein in the RER either remains in RER or localizes to other RER subdomain. It also can either target from ER to post ER compartment in endomembrane system Co-Translational Translocation of Soluble Protein into the RER Lumen Step 1  In cytosol translation of mRNA on free ribosome begins  N terminus of growing polypeptide emerges from ribosome and contains a signal sequence which serve as target towards ER  Exposed signal sequence is recognized by signal recognition particle (SRP)- has 6 proteins and 1 small RNA  SRP binds to ribosomes and stops translation Step 2 6  SRP targets complex (mRNA, polypeptide, and ribosome) to ER surface and binds to SRP receptor  Receptor is an integral membrane protein complex  Cytosolic domains of receptor serve as docking site for SRP Step 3  SRP released (back to cytosol) from receptor and at the same time ribosome binds to cytosolic side of translocon  Release step relies on GTP hydrolysis causing conformational change in SRP and receptor o Translocon=multiprotein complex that has many integral membrane protein subunits that form hour glass shaped aqueous pore. Aqueous pore contains pore ring that act as a gate o In closed state- pore ring opening too narrow for translocon and polypeptide o Opening is blocked by a plug (on ER side) which prevents movement of ions and small molecules between ER lumen and cytosol  Binding of ribosome to translocon results in continuation of translation  Signal sequence interacts with interior of translocon and changes conformation of translocon subunits, opening pore rings and removal of plug Step 4  Growing polypeptide moves through translocon and as signal sequence enters lumen it is cleaved by signal peptidase-faces ER lumen  As cleaved protein enters lumen it is glycosylated (addition of sugars) and is properly folded by reticuloplasmins (chaperones in ER)  Ribosome is released after transolaction and termination of translation and is returned to cytosol  Translocon closes pore ring and plug is readded Co Translational Insertion of an Integral Membrane Protein into RER  Most membrane proteins are also synthesized on membrane bound ribosomes on RER  Translocated during translation into the ER membrane in a similar manner to the import of a soluble protein of the ER lumen- there are a few differences resulting in mature protein being inserted into the ER membrane Step 1  N terminus of growing polypeptide enters translocon. The growing proteins first or only transmembrane domain (TMD) enters the interior of the translocon  TMD serves as a stop transfer sequence- interaction of hydrophobic TMD with hydrophobic pore ring stops and further translocation through translocon Step 2  Next the translocon opens laterally from the previous signal and the TMD segment of the protein is released laterally into membrane lipid bilayer  Cell wants positive charge on lumen side so n terminus must stay in lumen Step 3  Synthesis of proteins cytosolic facing C terminus resumes, the final orientation is N in lumen and C in cytosol Step 2a and 3a  Translocons interior interacts with proteins TMD to stop translocation and with several +ve changed amino acid residues located upstream (n terminal) of TMD  +vely charged amino acids determine topology  positive outside rule- TMD is re-oriented by the translocon so that the positively charged residues face the cytosol, and is released laterally into membrane lipid bilayer Step 4a  synthesis of the proteins C terminus resumes  final membrane orientation, N in cytosol and C in lumen **net positive charge must always be on the cytosolic side, net +ve change interacts with –ve charge on translocon from amino acids 7 Maintenance of Membrane Asymmetry Two Mechanisms 1. Lipid composition- inner and outer leaflet 2. Modification and orientation of integral membrane proteins (IMPs)- luminal domain of IMPs stays in lumen of compartments and forms an extracellular domain, the cytosolic domain is always in cytoplasm Membrane Biosynthesis in the ER  Membranes do not form de novo- they arise from pre existing ones  Most membrane proteins and lipids are made in ER but glycolipids (in golgi) and chloroplast and mitochondrial protein and lipids  Nascent ER membrane proteins and lipids may go to other ER domains or downstream organelles  Each organelle has unique complement of membrane protein and lipids  Membrane proteins and lipids are oriented in lipid bilayer in asymmetric manner  Integral membrane proteins- diff regions face cytosol and or lumen  Peripheral membrane proteins- on either cytosolic or luminal side of ER membrane  Membrane phospholipid- unequally placed between cytosolic and luminal leaflets of bilayer  Asymmetry is maintained throughout endomembrane system Processing of Newly Synthesized Proteins of ER  ER is the ideal processing site for nascent proteins- first compartment of endomembrane system  Final steps in cotranslational translocation pathway involve processing of new protein in ER lumen 1. Signal sequence cleavage- removal of N terminal signal sequence (signal peptidase) 2. Initial stages of glycosylation- additions of carb side chains to specific a.a’s of protein (glycoproteins are needed for proper folding and protein-protein binding) 3. Protein folding and assembly- protein folded by chaperones (reticuloplasmins) into proper 3D shape 4. Quality control- misfolded proteins are recognized and degraded Glycosylation of Proteins in the ER  Glycosylation- adding oligosaccharide chains to proteins forming glycoproteins, order of monomers in oligosaccharide is important  Functions of oligosaccharides on proteins- helps binding with other macromolecules, helps protein folding, needed in intracellular trafficking-traffics protein to destination  Most proteins made in ER are glycoproteins-proteins linked by a.a’s  Most common type of glycosylation N-linked (terminal amino group of asparagine N)  Two stages in N linked glycosylation: 1) core glycosylation (2) core modification- may occur or continue in Golgi Core Glycosylation-first stage  13 steps, various ER membrane bound glycosyltransferases synthesize core oligosaccharide  core= highly branched oligosaccharide chain containing 14 sugar residues and 3 glucose terminal branch  each enzyme (one at time) adds specific sugar  final step involves a glycosyltransferase linking core oligosaccharide to specific N residue on soluble or inte
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