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BIOLOGY 2B03 (285)
Kim Dej (39)
Lecture 5

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Department
Biology
Course
BIOLOGY 2B03
Professor
Kim Dej
Semester
Fall

Description
LECTURE 5: Part 1: Protein processing and folding Objectives;  Describe the structure of the RER  Describe protein modifications that occur in the RER  Describe the process of the unfolded protein response Endoplasmic reticulum (ER) in cultured epithelial cell: Immunofluorescence Green = ER  Single protein specific to the ER  Can highlight the ER specifically using antibodies Phase contrast:  All the membranes of the cell are visible  Plasma membrane and organelle membrane shown Live image:  Nucleus in blue  ER in red  Mitochondria in green/yellow  Undergo fission and fusion  Membranes are very dynamic and undergo constant change in structure 2 Components of the Endoplasmic Reticulum: 1. Rough Endoplasmic Reticulum (RER)  Studded by ribosomes attached to the membrane of the ER  Transport occurs between the RER and the Golgi apparatus o Proteins synthesized in the RER are moved via vesicles to the Golgi apparatus  The RER and Golgi target new proteins to different destinations o Part of co-translational pathway (or secretory pathway); where proteins are synthesized in the ER and go to components of the cell o Referred as secretory pathway because secreted proteins and antibodies are going to leave the ER, go through the Golgi apparatus and then be secreted out of the cell 2. Smooth Endoplasmic Reticulum (SER)  No ribosome association with the membrane  Protein synthesis does NOT occur  Synthesis of fatty acids and phospholipids occurs  Carbohydrates are metabolized  Calcium concentration is regulated Post-Translational Modifications and Quality Control: Modifications; 1. Glycosylation: covalent addition of carbohydrates  Addition of carbohydrate moieties to a protein  Primarily found on proteins that are secreted or plasma membrane proteins that have exoplasmic domain proteins  There are 2 types of glycosylation events; 1. N-linked  Most common form of glycosylation  Addition of oligosaccharide ‘block’ to 2H group of Asn 2. O-linked  Addition of 1-4 sugar residues to OH group of Ser, Thr  Modification occurs in the lumen of the ER o During the process of translation and transport through the membrane; a glycosyl group is being added  The oligosaccharide is added and attached to a transmembrane protein and is then transferred over to the protein which is being synthesized o Orientation of the protein is being maintained as it moves from the ER to the exterior of the cell o The orientation of the polarity of the glycosyl groups is being maintained  Glycosyl groups are added in the ER and stay within the interior of vesicles which transport to the Golgi apparatus  As the proteins travel to the next vesicle; the glycosyl groups are inside the membrane (not exposed to cytosol)  When proteins reach the exterior membrane the glycosyl groups are on the exterior of the cell (at no time do the glycosyl groups face the cytosol)  Functions of glycosylation; 1. Proper folding 2. Trafficking 3. Stability 4. Cell-cell adhesion 5. Cell identity 2. Disulphide bond formation  Cysteine bridges  Occur specifically in an oxidizing environment (occurs spontaneously in oxidizing environment) o ER is an oxidizing environment o Cytoplasm is a reducing environment (no spontaneous formation)  Primarily proteins that pass through the ER contain disulphide bonds  Protein Disulphide Isomerase (PDI): o Able to facilitate or accelerate the formation of disulphide bridges by producing an intermediate which interacts with 2 cysteine residues along a single protein (or between 2 different proteins) o Useful for correcting the formation of disulphide bonds;  As protein undergoes co-translational transport the first pair of cysteine residues that leave the ribosome that are transported through that channel into the lumen of the ER might form a cysteine bridge 3. Folding (before transport)  Accelerated by resident ER proteins; 1. Disulphide isomerases (S-S) 2. Chaperones (Hsp- BiP) 3. Lectins (carbohydrate binding proteins, calnexin, calreticulin) bind unfolded or misfolded polypeptides (via an N-linked oligosaccharide on polypeptides) 4. Peptidyl-prolyl isomerase (mediate rotation about peptide bonds)  Promote rapid and reliable folding of the protein (get protein to its native conformation) 4. Proteolytic cleavage (cleavage of peptide backbone of the protein)  Removal of signal sequences: o Secreted and Type I integral membrane proteins signal is cleaved by signal peptidase o Some proteins must be cleaved for proper folding to occur Quality control; 1. Unfolded protein response (UPR)  Process that makes sure proteins are folded before they leave the ER and go to their next destination  Mechanism of quality control (where unfolded proteins are protected and given time and tools to fold properly)  Excess unfolded protein in the lumen of the ER leads to: o Activation of UPR, proteins that make up the UPR include;  Hsp-BiP70; functions to act as chaperone (facilitate proper folding) and to activate the protein response  BiP will preferentially bind to the Ire1 monomer rather than the unfolded proteins. However, if there is a dramatic increase in the concentration of the proteins in the lumen of the ER then the BiP will overall have a preferential binding to the unfolded proteins (based on concentration levels). This results in the dimerization of the Ire1 monomer; which activates the Ire1 dimer (endonuclease – an enzyme with a specific function in the cell; has the ability to cut a nucleic acid [Hac1 mRNA, which is a transcription factor for proteins; code for ER lumen chaperone, which code for lectins, which code for PDI etc.]) o Reasons for excess unfolded proteins;  Cells making lots of secreted proteins, need extra chaperones  Cell has been heat stressed, proteins denature o What happens to unfolded/misfolded proteins in the ER?  Studies show that they are transported into the cytoplasm backwards through the Translocon  Ubiquitinylating enzymes localized on the cytosolic face of ER, ubiquitinate unfolded/misfolded proteins  Ubiquitinylated proteins targeted to the Proteasome Part 2: Protein trafficking via vesicles Objectives:  Describe the steps in vesicle formation and deposition  Describe and interpret the experiments used to create and test the models of vesicle transport Vesicles in protein transport:  While some proteins are resident in ER and Golgi (broken into the cis, medial and trans cisternae), others are targeted to the plasma membrane or to the lysosome (from the trans cisternae) in vesicles  Other proteins leave the cell, secreted via secretory vesicles = exocytosis  Vesicle transport is also involved in endocytosis – bringing components into the cell via membrane bound endosomes Golgi Complex:  After the ER transport goes to the Golgi complex (dynamic organelle)  Contains a series of flat membrane sacs (cisternae) and associated spherical vesicles The trans-Golgi network (TGN) where vesicles are leaving the Golgi The cis-Golgi network (CGN) is a composition of vesicles and sacs that are evolving DIC/Nomarski image:  Can see the membranes that are making up the cell  Can see nucleus in the middle  Can see plasma membrane on the outside  How do we know which membranes are specifically a part of the Golgi apparatus? o Wheat germ agglutin is used rather than an antibody o It is able to bind to the N-linked polysaccharides (which are found within the Golgi apparatus) o In this case; the agglutin is fluorescently-labelled so we can see specifically the Golgi apparatus and not the membranes of the ER or other organelles Four Steps to Vesicular Trafficking: Transport of membrane and soluble proteins between membrane bound compartments is mediated by transport vesicles; 1. Vesicles form by
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