Chapter 8

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Biological Sciences
David Harrison

Chapter 8 Cell Biology Cytoplasm of a cell is divided into organelles, they are dynamic compartments. Endoplasmic Reticulum, Golgi complex, endosomes, lysosomes and vacuoles all part of endomembrane system. 8.1 An Overview of the Endomembrane System Organelles of endomembrane systems is a network in which materials are shuttled back and forth. Materials are shuttled between organelles from Golgi to plasma membrane. Done in small transport vesicles. Transport vesicles move through cytoplasm in a directed manner, often pulled by motor proteins on tracks formed by microtubules. At destinations, they fuse with membrane of acceptor compartment. Several pathways: Biosynthetic pathway – proteins are synthesized in the endoplasmic reticulum, modified, through the Golgi complex, and transported to various destinations. Also referred to as: Secretory pathway as proteins are synthesized and secreted. Secretory activities of cell include: Constitutive secretion – materials are transported in secretory vesicles from their sites of synthesis and discharged into the extracellular space. Regulated secretion – materials are stored as membrane bound packages and discharged only in response to an appropriate stimulus. Some materials are stored in secretory granules. Proteins, lipids and complex polysaccharides are transported through cell along secretory pathway. Soluble proteins are discharged, integral proteins of the various membrane, and soluble proteins that reside within the various compartments. Endocytic pathway, materials move from the outer surface of the cell to compartments, endosomes an lysosomes. Different organelles contain different integral membrane proteins. All of the proteins and cargo around the cell know where to go because of sorting signals. They are recognized by receptors that reside in membranes of budding vesicles. 8.2 A few Approaches to the Study of Endomembranes Insights Gaines from Autoradiography Acinar cells of pancreas have extensive endomembrane system. Function primarily in synthesis and secretion of digestive enzymes, after secretion enzymes are shipped through ducts to small intestine then they ingest the food. Autoradiography provides a means to visualize biochemical processes by allowing an investigator to determine location of radioactively labelled materials within a cell. Pulse Chase – incubating the tissues for a brief period in radioactive amino acids, washed tissue free of excess isotope and transferred tissues to a medium containing only unlabeled amino acids. Pulse refers to brief incubation with radioactivity during which labelled amino acids are incorporated into protein. Chase refers to the period when tissue is exposed to the unlabeled medium. Insights Gained from the Use of the Green Fluorescent Protein Green Fluorescent Protein (GFP) emits a green fluorescent light. GFP is fused to DNA to be studied and the resulting chimeric is introduced into cells. Insights Gained form the Biochemical Analysis of Subcellular Fractions Techniques to homogenize or break up cells and isolate parituclar types of organelles. Vesciles derivd from different organelles have different properties which allow them to be separated form one another through subcellular fractionation. Membrane vesicles from endomembrane system form heterogeneous collection of similar sized vesicles called microsomes. 8.3 The Endoplasmic Reticulum Endoplasmic Reticulum is divided into Rough Endoplasmic Reticulum, and Smooth Endoplasmic Reticulum. The composition of the luminal, or cisternal space inside the ER membranes is quite different from that of the surrounding cytosolic space. Rough ER has ribosome’s bound to its cytosolic surface, RER is typically composed of a network of flattened sacs (cisternae), it is continuous with the outer membrane of the nuclear envelope. SER membranous elements are typically tubular and form interconnecting system of pipelines curving through cytoplasm. SER fragments into smooth surfaced vesciles, RER fragments into rough surfaced vesicles. Smooth Endoplasmic Reticulum SER is developed in skeletal muscle, kidney and steroid producing endocrine glands. Functions: - Synthesis of steroid hormones. - Detoxifaction in the liver. - Sequestering calcium ions. Funtions of the Rough Endoplasmic Reticulum The Rough ER is the starting point of the biosynthetic pathway: site of synthesis of the proteins, carbohydrate and phospholipids. Synthesis of Proteins on Membrane Bound versus Free Ribosomes Polypeptides are synthesized at two distinct locales within the cell. 1. Certain polypeptides are synthesized on ribosomes attached to the cytosolic surface of the RER membrane. Secreted proteins, integral membrane proteins, soluble proteins reside in compartments of endomembrane system. 2. Other polypeptides are synthesized on free ribosomes. Proteins destined to remain in Cytosol. Peripheral proteins. Proteins transported to the nucleus. Proteins to be incorporated. Signal hypothesis 1. Secretory proteins contain a signal sequence at their N terminus that directs the emerging polypeptide and ribosome to the ER membrane. 2. Polypeptide moves into the cisternal space of the ER. Moves through membrane as it is being synthesized. Synthesis of Secretory, Lysosomal, Or Plant Vacuolar Proteins on membrane Bound Ribosomes. Synthesis of polypeptide begins after mRNA binds to a free ribosome. Taken from the same population pool as those used for production of proteins that remain in Cytosol. They include hydrophobic amino acid residues. A nascent polypeptide is one in the process of being synthesized. Signal Recognition Particle (SRP) which onsits in mammalian cells of six distinct polypeptides and a small RNA molecule, called 7S RNA. SRP binds to both signal sequence on nascent polypeptide and the ribosome. Bound SRP serves as a tag that enables it to bind specifically to the cytosolic surface of the ER membrane. Binding to ER takes 2 things, one between SRP and the SRP receptor, and the other between the ribosome and the translocon. Translocon is a protein lined channel embedded in ER. Once the SRP ribosome nascent chain complex binds to the ER membrane, SRP is released from its ER receptor, the ribosome becomes attached to the cytosolic end of the translocon, and the signal sequence on the nascent polypeptide is inserted into the narrow aqueous channel of the translocon. The growin polypeptide is then translocated through the hydrophobic pore ring and into the ER lumen. The pore expands. The membrane bound ribosome is released from the ER membrane and the helical plug is reinserted into the translocon channel. Regulated by the binding or hydrolysis of GTP. GTP binding play key regulatory roles in different cellular processes. G proteins act liek molecular switches, GTP bound protein typically turns the process on and hydrolysis of the bound GTP turns it off. SRP and receptor are G proteins. Processing of Newly Synthesized Proteins in the Endoplasmic Reticulum The N terminal portion containing the signal peptide is removed from the most nascent polypeptides by a proteolytic enzyme signal peptidase. Carbohydrates are added by enzyme olgiosaccharyltransferase both are integral membrane proteins. Protein disulfide isomerise, Proteins enter the ER lumen with their cysteine residues in the reduced (-- SH) state, but they leave compartment with many of these residues joined to one another as oxidized sulphides. Formation is catalyzed by PDI. Play important role in maintain stability of proteins. Endoplasmic reticulum is ideally constructed for its role as a port of entry for the biosynthetic pathway. Membrande provides a large surface area. Lumen of ER provides a local environment. Synthesis of Integral Membrane Proteins on Membrane Bound Ribosomes Integral membrane proteins are also synthesized on membrane bound ribosomes of the ER. The use same machinery. Integral proteins contain one or more hydrophobic transmembrane segments that are shunted directly from channel of translocon into lipid bilayer. These segments of the nascent polypeptide that are sufficiently hydrophobic will spontaneously dissolve into the lipid bilayer and ultimately become transmembrane segments of an integral membrane protein. Single spanning membrane proteins can have an orientation with their N terminus facing either the Cytosol or the lumen of the ER. That a translocon, by itself is capable of properly orienting transmembrane segments. Translocon is a complex machine capble of recognizing various signal sequences. Membrane Biosynthesis in the ER Membranes arise from pre-existing membranes. Membrane components move from the ER to virtually every other compartment tin the cell. Its lipids and proteins are modified by enzymes, modifications contribute to a unique composition. This asymmetry is established initially in the endoplasmic reticulum. Asymmetry is maintained as membrane carriers bud from one compartment and fuse to the next. Synthesis of Membrane Lipids – Most membrane lipids are synthesized entirely within the endoplasmic reticulum.Some of these lipid molecules are later flipped into the opposite leaflet through the action of enzymes called flippases. Membranes of different organelles have different lipid composition. Several factors contribute to changes. 1. Most membranous organelles contain enzymes that modify lipids already present. 2. When vesicles bud some types of phospholipids may be preferentially included while other types are left behind. 3. Cells contain phospholipid transfer proteins that can bind and transport phospholipids through the aqueous Cytosol from one membrane compartment to another, movement of specific phospholipids from the ER to other organelles. Glycosylation in the Rough Endoplasmic Reticulum Nearly all of the proteins produced on membrane bound ribosomes become glycoproteins. Carbohydrates groups have key roles they also aid in the proper folding of proteins. The sequences of sugars are highly specific. The addition of sugars to an olgiosaccharide chain is catalyzed by a family called glycosyltransferases. These enzymes transfer a specific monnosccharide from a nucleotide sguar to the growing end of the carbohydrate chain. Arrangemnt of sugars depends on the spatial localization of particular enzymes in the assembly line. Carbohydrate chain is not assembled on the protein itself but put together independently on a lipid carrier and then transferred to a specific asparagines residues. This lipid carrier dolichol phosphate, is embedded in the ER membrane. Sugars are added one at a tyme by glycosyltransferases. Mutations that lead to the total absence of N glycosylation cuase the death of embryos prior to implantation. From the ER to the Golgi Complex: First step in vesicular transport Exit sites of the RER cisternae are devoid of ribosome’s and serve as places where the first transport vesicles in the biosynthetic pathway are formed. Transport vesicles fuse with one another to form larger vesicles and interconnected tubules in the region between the ER and Golgi complex. ERGIC endoplasmic reticulum Golgi intermediate compartment and the vascular tubular ca
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