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Intracellular Compartments and Protein Sorting The Endoplastic Reticulum Pages 727-745 3 Main Functions of the ER 1. Site of protein synthesis -Transmembrane proteins -Water-soluble proteins (cell exterior) 2. Site of lipid biosynthesis for most of the organelles 3. Storage of calcium in the ER lumen EM of Rough ER Pancreatic exocrine cell Smooth ER -a point where vesicles bud from the ER in transport to the Golgi -Prominent in cells which specialize in lipid metabolism, synthesize steroid hormones, and in cells involved in detoxification reactions Isolation of Rough and Smooth Microsomes from the ER Purified Rough ER Fraction Co- and Post-Translational Protein Translocation Free and MembraneBound Ribosomes Co-translational process The Signal Hypothesis -Signal Sequences were first discovered in the 1970s in ER proteins -The signal hypothesis was tested by genetic and biochemical experiments Signal-Recognition Particle -SRP guides the ER signal sequence to the ER membrane and binds with the SRP receptor -SRP homologues are found in all organisms that have been studied 6 Proteins Signal-Sequence Binding Pocket -ER signals vary in amino acid sequence but each has at least 8 nonpolar amino acids -The binding pocket is a large hydrophobic pocket lined by methionines -methionines can accommodate sequences of different size and shape ER Protein Targeting -An ER Signal is recognized twice Ribosome bound to the Sec61 Translocator When a protein is not transversing the pore, either a lumenal ER protein serves as a plug or the translocator itself can close the pore 3-4 Protein Subunits creates a water-filled pore Continuous Pore Joining of the ER Lumen and the Ribosome 3 Methods of Protein Translocation Chaperone Proteins prevent the protein from folding in the cytosol 20 AA / ATP Soluble Protein Translocation The ER signal sequence alone is not sufficient For signal cleavage, an adjacent cleavage site is needed The ER signal sequence has 2 functions: 1. it directs the protein to the ER and 2. is serves as a start-transfer signal Single-Pass Transmembrane Protein Hydrophobic sequence Transmembrane Protein with an Internal Signal Sequence, Method 1 Orientation of the start-transfer sequence Determines which protein segment is moved across the membrane into the ER lumen Transmembrane Protein with an Internal Signal Sequence, Method 2 Double-Pass Membrane Protein All copies of the same protein will have the same orientation in the lipid bilayer creating an asymmetrical ER membrane Multipass Membrane Protein Ex. Rhodopsin in rod photoreceptor cells ER Resident proteins -They contain an ER retention signal of four amino acids At their C terminus that is responsible for retaining the protein in the ER Return to ER signal -Lys-Asp-Glu-Leu-COO– Protein disulfide isomerase (PDI) catalyzes the oxidation of free SH groups on cysteines to form S-S bonds. All proteins in the lumen of the ER and other organelles and the extracellular space are disulfide-bonded, while those in the cytosol are not N-Linked Glycosylation -Glycoproteins – proteins containing sugar residues -Linked to Asparagines in the sequences Asn-X-Ser and Asn-X-Thr, where X cannot be Pro -Transfer is catalyzed by oligosaccharide transferase which has its active site on the lumenal side of the ER Protein Glycosylation The precursor oligosaccharide is held in the ER membrane by a long and very hydrophobic lipid molecule, dolichol Precursor Oligosaccharide Synthesis -The sugars are first activated in the cytosol by forming nucleotide-sugar intermediates -O-linked oligosaccharides are linked to hydroxyl groups of Ser and Thr residues and this occurs in the Golgi Glycosylation in Protein Folding Calreticulin and calnexin both require calcium Degradation of Misfolded ER Proteins Retrotranslocation or dislocation occurs via the same translocator complex, Sec61 Unfolded Protein Response in Yeast GPI Anchor Attachment to Proteins The protein can be released through cleavage by a specific phospholipase GPI - glycosylphosphatidyl-inositol Lipids and Membrane Components Phosphatidylethanolamine Phosphatidylserine Phosphatidylcholine Cholesterol Ceramide (Sphingomyelin) Glycolipids Synthesis of Phosphatidylcholine Occurs exclusively in the cytosol This step enlarges the lipid bilayer Role of Phospholipid Translocators Phospholipid Exchange Proteins -These are water-soluble proteins which transfer individual phospholipid molecules between membranes -PC and PS are imported into the Mito this way and then some of the PS is converted to PE