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BSC 300, Week 8

by: Ashley Bartolomeo

BSC 300, Week 8 BSC 300

Ashley Bartolomeo
GPA 3.9

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Notes on chapter 8, first half
Cell Biology
John yoder
Class Notes
Cell, Biology
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This 8 page Class Notes was uploaded by Ashley Bartolomeo on Wednesday October 5, 2016. The Class Notes belongs to BSC 300 at University of Alabama - Tuscaloosa taught by John yoder in Fall 2016. Since its upload, it has received 4 views. For similar materials see Cell Biology in Biology at University of Alabama - Tuscaloosa.


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Date Created: 10/05/16
Chapter 8 Cytoplasmic Membrane Systems: Structure, Function and Membrane Trafficking Key Concepts  Understand the dynamic nature of the endomembrane system within the cell  Discriminate between regulated and constitutive secretion  Elucidate the structure and function of the rough and smooth ER  Outline events in synthesis and transport of membranes/proteins through the cell  Elucidate role and sites of glycosylation in processing of secretory/integral membrane proteins  Elucidate the structure, function and polarization of the Golgi complex  Describe role of various types of coated-and non-coated vesicles in membrane trafficking  Explain the signals used to target proteins of exocytosis and its triggers  Describe the lysosomal structure/function and diseases caused by lysosomes malfunction  Distinguish between phagocytosis, bulk phase endocytosis and receptor-mediated endocytosis Introduction  Membranes divide cytoplasm of eukaryotic cell into discrete functional compartments  Endomembrane system includes membranes of the organelles: nucleus, endoplasmic reticulum, Golgi complex, endosomes, lysosomes, and vacuoles functioning as an interconnected, but discontinuous coordinated unit Overview of the Endomembrane System  Materials (principally proteins and lipids) are produced, modified and shuttled back and forth via membrane-bound transport vesicles  Some of this traffic includes molecules that will remain associated with the membranes of the various organelles, but in many cells a large part of this cargo, is protein bound for export via exocytosis Biosynthetic and Secretory Pathways  Biosynthetic or secretory pathway: synthesis, modification and transport of proteins and lipids through the endomembrane system for ultimate release from secretory cells o Constitutive secretion – performed by most cells: functions to maintain the extracellular matrix and plasma membrane o Regulated secretion – secretion occurs only in response to a specific stimulus  Examples: digestive enzymes of pancreatic cells, neurotransmitters and hormones  A coordinated targeting system is required to properly identify the correct destination for the newly synthesized proteins and lipids  Similarly, endocytosis of various extracellular requires sorting and delivery of these molecules to the appropriate destinations Study of Cytomembranes Autoradiography  Autoradiography- uses radioactively labeled materials and exposure to photographic film to analyze diverse biological processes  Combined with subcellular fractionation this was an invaluable in determining the order of protein synthesis and trafficking through the endomembrane system  Techniques utilizing autoradiography have largely been replaced by fluorescent-based approaches Overview of the Endomembrane System Synthesis and Transport of Secretory Proteins  Incubate cells with radioactively labeled amino acids (pancreatic acinar cells produce lots of protein) o Fix immediately and expose to film, majority of radioactivity localized to ER o Fixation after 17 minutes, majority of radioactivity found in Golgi apparati and a few deep vesicles o Fixation after 2 hours, majority of radioactivity found in apically localized excretory granules Study of Cytomembranes Biochemical Analysis of Subcellular Fractions  Homogenize cells and isolate organelles, which can be further separated from one another by subcellular fractionation techniques (centrifugation that separates organelles based on density, buoyance or mass)  Protein composition of the various membrane systems can be determined by mass spectrophotometry GFP-based Protein Tracking  Green fluorescent protein (GFP) – a small fluorescent protein isolated from a jellyfish  A GFP gene chimera allows observation of protein synthesis in the cell  Here the fusion protein harbors a temperature sensitive mutation that prevents export from the ER where it is synthesized when cultured at 40C  When shifted to 32C, the fusion protein folds properly, and is exported, allowing the researchers to monitor in real time the trafficking of the protein Genetic Mutants  Budding yeast Saccharaomyces cerevisiae has been a powerful genetic model system for identifying genes involved in vesicular transport and exocytosis  Mutation screens isolate mutants of genes necessary for specific processes in vesicle formation, trafficking or fusion  Cloning gene provides protein sequence and therefore indicates what specific function each protein may play Utility of Genetic Mutants  RNA interference: a genetic technique that blocks translation of a specific mRNA (generates a loss of function phenotype for the associated gene)  Can be used as a rapid screening approach to identify genes required for a specific cellular process The Endoplasmic Reticulum  A membranous network, continuous with the outer nuclear membrane, that penetrates into much of the cytoplasm  Like other organelles, the ER is highly dynamic undergoing continual turnover and reorganization  Divided into two sub compartments: o Rough endoplasmic reticulum (RER) o Smooth endoplasmic reticulum (SER)  Different types of cells have different ratios of the two ER domains, depending on activities of the cell Rough ER:  Composed of a network of flattened sacs called cisternae  Continuous with the outer membrane of the nuclear envelope  Principle role: synthesis of proteins to be part of the endomembrane system or exocytosed  Covered with numerous ribosomes on its cytosolic surface for this purpose  Recall, ribosomes are the complexes that translate mRNAs into protein Smooth ER:  Continuous with RER. Extensively developed in a number of cell types; functions include: o Synthesis of steroid hormones in endocrine cells: such as in the gonad and adrenal cortex o Liver: detoxification of various organic compounds through the actions of the Cyt P450 enzyme family o Sequestration of calcium ions from cytoplasm of muscle cells, which contains a high concentration of calcium-dependent proteins. Ca2+ release from SR highly regulated to control activity of these proteins Both rough and smooth ER are responsible for new lipid synthesis, but in cells with heavy protein production, and therefore extensive ribosomes on the RER, this job is primarily performed by the SER Synthesis of Proteins on Membrane-Bound Versus Free Ribosomes  ~1/3 of polypeptides encoded by human genome synthesized on RER ribosomes: secreted proteins, integral membrane proteins, and soluble proteins of organelles  Polypeptides synthesized on “free” ribosomes include: cytosolic proteins, peripheral membrane proteins, nuclear proteins, and proteins destined for chloroplasts, mitochondria and peroxisomes Free or RER Synthesis Determined by N-terminal Amino Acid Sequences  All translation begins in the cytoplasm on free ribosomes  N-terminus first end to be translated  Proteins destined for RER synthesis possess an N-terminal signal sequence  Which directs transfer of the ribosome/mRNA/peptide complex to the RER  Polypeptide is translated into ER cisternal space through a protein-lined pore  A small number of proteins have the signal sequence at the C- terminus, and are thus completely translated before they are delivered to the RER Synthesis of Proteins: A Review  Transcription and translation will be covered in lectures 20/21  mRNA transcripts, after being processed, are bound in the cytoplasm by free ribosomal small subunits  Once the initiation codon is recognized, the small subunit is joined to the large subunit and translation is initiated  The transcript is read from 5’ to 3’ direction as individual triplet codons of nucleotides  Translation proceeds through addition of individual amino acids that correspond to the individual codons  Peptides are synthesized from the N-terminus to the C-terminus Synthesis of Protein of Membrane Bound Ribosome Synthesis of secretory proteins on RER ribosomes  Messenger RNA is bound by free ribosome in cytoplasm  An N-terminal signal sequence on the nascent protein is recognized by a protein called the signal recognition particle (SRP)  SRP binds the signal sequence and a receptor protein on the RER surface, delivering the translation complex to the protein translocon complex that mediates RER protein translocation  Once in the RER lumen, the signal sequence is cleaved by signal peptidase  Chaperones (like Bip) assist in proper folding  Because the extracellular space is a reducing environment, such secrete proteins )and the extracellular portion of integral membrane proteins) are stregnthered by the generation of di-sulfide bridges – bonds catalyzed by protein disulfide isomerase in the RER Processing of Newly Synthesized Proteins in the ER  Most proteins produced in the ER and processed in the Golgi are extensively modified by oligosaccharide additions. Such carbohydrate additions are initiated in the ER by the enzyme oligosaccharyltransferase Synthesis of Integral Membrane Proteins on the RER  Integral membrane proteins contain hydrophobic trans-membrane domains that interfere with translocation through hydrophilic core of the translocon  X-ray crystallographic studies suggest the translocon is clam-shaped and oscillates between open and closed conformations  This allows stretches of amino acids with sufficient hydrophobicity to dissolve into the lipid bilayer  The translocon also assists in the proper orientation of transmembrane sequences, that is, will the N-terminus face the lumen or cytoplasm  In general, amino acids on the cytoplasmic side of TM domains are more positively charged than the luminal side – serves as a signal to the translocon for proper orientation (it can physically invert the TM domain)  The TM domains of proteins with multiple passes, in general, alternate in direction, therefore, it is the first TM domain that establishes the orientation of all others  For proteins with cytoplasmic N-termini, the signal peptide is often internal, so it is inserted into the translocon leaving the N-terminus projecting into the cytoplasm, thereby establishing the orientation of this and subsequent transmembrane domains Membrane Biosynthesis in the ER:  Recall membranes are often highly asymmetric with distinct lipids composition in each leaflet  This asymmetry is established in the ER and maintained as vesicles carry lipids and proteins through the endomembrane system  As these new membranes move from one compartment to the next, its proteins and lipids are extensively modified Modifying the Lipid Composition of Membranes  Most membrane lipids are synthesized entirely within the ER. Sphingomyelin and glycolipids require additional processing in the Golgi. Some unique lipids of mitochondria and chloroplasts are synthesized in those membrane systems  Enzymes hat catalyze phospholipid synthesis are associated only with the outer ER leaflet – facing the cytoplasm. As a result, all new phospholipid synthesis and integration occurs in the outer leaflet  Newly synthesized phospholipids are inserted into the cytoplasmic leaflet  Asymmetry is established through the action of: o Scramblases: evenly distribute lipids to both side of the membrane. non-specific and do not require input of energy o Flippases: ATP-dependent and lipid specific, catalyze uni- directional leaflet translocation. Responsible for establishing lipid asymmetry  Factors contribution to variation of organelle lipid composition o 1. Organelle – specific enzymes interconvert lipids o 2. Inclusion/exclusion process during vesicle formation o 3. Lipid-transfer proteins bind and transport lipids without the use of vesicle transport Synthesis of the Core Portion of an Oligosaccharide N-linked Glycosylation in the RER  Nearly all ER synthesized proteins are extensively modified via glycosylation as they traverse the endomembrane system  Such modification catalyzed by destination specific glycosyltransferase enzymes  Ultimate composition and arrangement of sugars (glycosylation) for any protein is both cell-type and organelle specific  Meaning that in different cell types, the same protein can have distinct glycosylation patterns  These modifications alter protein conformation, solubility, participate in inter- and intracellular signaling, protein-protein interaction and cell- cell recognition  All proteins originating in the endomembrane system are initially glycosylated by a core carbohydrate chain of 14 sugar monomers. It is assembled and covalently linked to the lipid carried dolichol phosphate  The composition and orientation of these sugar monomers is identical for all ER processed proteins: 2 NAG, 9 mannose and 3 glucose monomers assembled in a trident orientation  Assembly begins on the cytoplasmic face, the glycolipid is flipped to the ER lumen, and glycosylation is completed  Glycosyltransferase enzymes catalyse transfers via the hydrolysis of phosphorylated nucleotides covalently attached to the sugar monomers  Once assembled, this glycan is transferred to specific asparagine residues within the sequence Asp-X-Ser/Thr by the enzyme oligosaccharyltransferase  Because these new covalent bonds are formed between a sugar and the nitrogen within the side chain of asparagine, the process is referred to as N-linked glycosylation  A small number of O-linked proteoglycans are assembled, one sugar at a time, on serine or threonine residues (-OH) in the Golgi apparatus Quality Control: ERAD and the Unfolded Protein Response  After transfer to the polypeptide, the core oligosaccharide begins to be extensively modified  First part of this regulates a process that ensures proper folding of ER synthesized protein: called the ER quality control (ERQC) system  Enzymes called glycosidases cleave 2 of the three terminal glucose monomers and the monoglucosylated polypeptide is bound by a chaperone protein (either calnexin or calreticulin)  These chaperons promote proper folding by preventing aggregation and premature export from the ER  The remaining glucose residue is trimmed by a glucosidase  An enzyme named UGGT (UDP glucose/glycoprotein glucosyl transferase) inspects the new protein for proper folding (e.g., are there any exposed hydrophobic residues?)  If the protein is folded correctly it is released and sequestered for export in vesicles  If the protein is misfolded, UGGT monoglucosylates it on the core glycan and it is again bound by ER chaperone proteins  Several such rounds may occur before the protein is finally released for export, or recognized as defective ER-associated degradation (ERAD)  Improperly folded proteins are transferred to membrane associated ubiquitin ligases, which add long chains of the protein ubiquitin to these aberrant proteins  Such tagged proteins are exported to the cytoplasm where they are digested by proteasomes  This, for example, is the fate of the CFTR proteins that fail to fold properly in people with cystic fibrosis The Unfolded Protein Response (UPR)  Under controls of extreme stress, misfolded proteins can be generated faster that the ERAD system can process them  This triggers the UPR: o 1. Halt protein translation to prevent accumulation of misfolded proteins o 2. Degrade misfolded proteins o 3. Activate signaling pathways that lead to increased production of ER chaperones o 1. Membrane associated protein sensors that relay information about misfolded proteins are normally kept in an inactive state, through association with chaperones proteins called BiP o 2. Accumulation of many unfolded proteins sequesters the BiP chaperones, thus releasing and activating the sensors o 3. Released sensors activate different parts of the UPR:  A. One sensor funtions as a kinase that phosphorylates and inactivates a key protein involved in protein translation (elF2a). this inhibits protein synthesis and reduces accumulation of new proteins  B. Another sensor translocates to the nucleus where it activates expression of several genes that participate in the response (chaperones, vesicle coat proteins etc)  Prolonged UPR activation signals that the cell will not be able to resolve the misfolded protein challenge  Accumulation of the activated sensors leads to stimulation of the programmed cell death pathway (apoptosis) – a pathway whose details we’ll revisit later in the semester From the ER to the Golgi Complex: The First Step in Vesicular Transport  RER has specialized exit sites where transport vesicles are formed and released (no ribosomes)  Transport vesicles fuse with one another and form the ERGIC (endoplasmic reticulum Golgi intermediate compartment) – a shuttle system that delivers proteins and newly synthesized lipids to the Golgi complex for further processing and sorting  Movement of these vesicles is mediated by motor proteins trafficking along microtubules


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