E2 - ER & Ribosomes

Describe the following functions associated with the ER: 1) Phospholipid, Triglyceride, and Cholesterol Synthesis

Phospholipid, Triglyceride, and Cholesterol Synthesis: Most all produced by integral membrane proteins on cytosolic side of SER (some on RER). 1) Phospholipid Synthesis: Fatty acids with CoA and glycol-3-phosphate placed in membrane by acyl tranferase. Phosphatase then removes phosphate and cholinephosphotransferase sticks a phosphatidylcholine on it = Phospholipid 2) Triglyceride Synthesis: Fatty acids with CoA and glycol-3-phosphate has its CoA removed then placed in ER membrane. Phosphatase removes phosphate and replaces it with -OH. Acetyltransferase adds another fatty acid to chain by removing its CoA = TRIglyceride. 3) Cholesterol Synthesis: Made in ER; acetate to cholesterol. HMG-CoA reductase is principle step in its biosynthesis.

Compare similarities and differences between procaryotic and eucaryotic ribosomes. (composition, size, polymerases, etc.)

Procaryotic Ribosomes: a. 70s (50s & 30s) b. Has only 1 type of RNA polymerase. c. Transcription and translation are simultaneous. Eucaryotic Ribosomes a. 80s (40s & 60s) b. Large due to production of more protein. c. 3 different types of RNA polymerases: (1) Ribosomal RNA transcribed by RNA polymerase I (2) mRNA transcribed by RNA pol II (3) tRNA transcribed by RNA pol III.

1) Describe the interconnections between ER and nuclear membrane. 2) Discuss the fate of the endoplasmic reticulum when the cell is homogenized.

1) a. There's a space where nuclear membrane and ER connect. b. ER is continuous with outer nuclear membrane. c. Relationship is not completely clear. 2) ER spreads through entire cytoplasm, so cannot isolate the ER as an intact organelle. When cell homogenized, ER is broken into vesicles called microsomes. - Microsomes can be purified by centrifugations & various sucrose gradients. - Ribosomes can then be stripped off of RER for independent study.

Describe in detail: 1) Association of ribosomes with rough endoplasmic reticulum (RER) 2) Process of protein synthesis on RER. 3) The "Signal Hypothesis" by Blobel

1) Association: Ribosomes (250Å) studded in circular polysomes along cytosolic surface of RER are producing proteins intended for transport out of cell or proteins intended for membranes. (polysomes = cluster of ribosomes) 2/3) RER Protein Synthesis & Signal Hypothesis: a. Initiation: Synthesis of proteins targeted for ER is initiated by binding of mRNA molecules to free ribosomes in cytosol. mRNA determines what type of protein any given ribosome will make and where the protein will eventually reside. (Prior to mRNA binding, ribosomes are all functionally equivalent.) b. Binding of SRP: On mRNA, the AA's that are initially linked together for secretory protein form hydrophobic stretch called signal peptide that is first part of protein, exiting pore on large subunit. It's then recognizable by SRP component (w/ hydrophobic crevice) and binds to it. c. Attachment of ribosome to ER: Mediated by receptor for the SRP on ribosome, SRP-ribosome complex then docks on translocation pore that sits in the ER & signal peptide binds to receptor on ribosome. d. Cleavage of signal sequence: GTP binds to SRP and receptor, unblocking translation. ER pore opens and N-terminus (with signal) is inserted into protein-conducting channel (pore). GTP is hydrolyzed and SRP is released. Inside, signal peptidase (protease) clips off signal peptide at the point when cleavage site of protein is exposed. e. Completion of protein: After signal sequence removed, translation continues. Protein released into ER lumen and it's modified into mature protein. Eventually viscles bud off in golgi. Golgi fuse to membrane and protein is secreted. Cytoplasmic proteins don't have signal peptides (target mechanism).

Describe the following functions associated with the ER: 1) Calcium Storage and Release 2) Membrane Flow & Mechanical Support

1) Calcium Storage and Release: Ca is stored in Smooth ER of muscle cells. Ca ions pumped into ER by ATPases and are stored in SER on Ca binding proteins. The binding of neurotransmitter molecules to receptors on surface of muscle cell triggers a signal cascade that leads to release of Ca and contraction of muscle fibers. 2) Membrane Flow and Mechanical Support: - Membrane Flow: ER → Golgi → secretory vesicles. Membrane thickness increases as flow travels towards PM. RER = 5nm thickness while PM = 8nm thickness. - Mechanical Support: Adds mechanical strength along with cytoskeleton since it pervades most of cytoplasm.

Explain how quality control is applied to proteins for export: 1) Why is quality control needed? 2) What is/are used to meet these needs?

1) Correct protein folding essential for life. If incorrect folding, proteins cannot be exported from ER. Stress may also cause unfolding. To keep this from happening, Molecular Chaperones are used for quality control. Accumulation of unfolded proteins cell respond by a decrease in rate of protein synthesis and an increase amount of chaperones. 2) Chaperones: Proteins that facilitate correct assembly of proteins but are not components of assembled structures. a. They function by binding to specific regions that are exposed only in early stages of assembly, thereby inhibiting unproductive assembly pathways that would lead to incorrect structures.

Describe the following functions associated with the ER: 1) Detoxification of Compounds 2) Glycogen Metabolism

1) Detoxification of Compounds: Smooth ER (mainly in liver) detoxifies substrates by: a. Oxidation of substrates b. Reduction of substrates c. Conjugation of substrates d. Hydroxylation of substrates (cyt P450) - Hundreds of these, each one specific to a substrate. P450 takes e- from NADH or NADPH via the ETC and uses it to hydrolyze O2. 1 O goes to water and other to hydrolyze the substrate. *Main point is to make substrate water soluble so it can be excreted from body. Occasionally substrates can combine to become more soluble.* 2) Glycogen Metabolism: Smooth ER in liver also involved in enzymatic breakdown of stored glycogen by glucose-6-phosphotase. This enzyme is anchored to ER & contains structures allowing removal of phosphate & free glucose.

Discuss the role of: 1) HMG-CoA Reductase in cholesterol synthesis 2) Statin drugs on this process.

1) HMG-CoA Reductase: A critical enzyme for cholesterol biosynthesis in liver. 2) Statins: a. HMG-CoA reductase inhibitors, lowering cholesterol levels. b. Competitive inhibitors (similar in structure) by taking the place of HMG-CoA in enzymes and reduce rate of mevalonate production (the next molecule in cascade that eventually produces cholesterol).

Describe protein glycosylation and the role dolichol phosphate plays. 1) Give 5-Step Process

1. Glycosylation begins as dolichol phosphate (DP), an oligosaccharide carrier, is inserted into the ER membrane. N-acetyl glucosamine and mannose groups are added to phosphate group of DP. 2. Growing core oligosaccharide then translocated from cytosol to ER lumen by phospholipid translocator (flippase). 3. Once inside ER lumen, more mannose and glucose units are added. 4. Completed core oligosaccharide then transferred as single unit from dolichol to N-terminal of lysine or asparagine residue of recipient protein (usually while peptide being synthesized by ER bound ribosome). 5. Core oligosaccharide attached to protein is trimmed & modified.

Describe the general structure and composition of ribosomes.

Composition: a. Made of RNA and proteins, in almost same amount. b. RNA derived from nucleus, where ribosomes are synthesized in a cell. c. Single cell prokaryotes (i.e. bacteria) composed of some thousands of ribosomes, while a highly developed eukaryotic cells (i.e. human cells) have several million. d. Prokaryotic ribosomes are smaller than eukaryotic. Structure: a. Typically consists of 2 subunits, each containing RNA and proteins. b. Both subunits classified with respect to sedimentation rate in particular environment.

List the components necessary for protein synthesis and explain their function.

Ribosome: A & P sites loaded with tRNAs that bring AA's together as mRNA goes through. Read: The DNA housed in the nucleus is too large to move through the nuclear membrane, so it must be copied by the smaller, single-stranded RNA (transcription), which moves out of the nucleus to ribosomes located in the cytoplasm and rough endoplasmic reticulum to direct the assembly of protein (translation). The genes do not actually make the protein, but they provide the blueprint in the form of RNA, which directs the protein synthesis. Transcription occurs in the cell nucleus and represents the transfer of the genetic code from DNA to a complementary RNA. The enzyme RNA polymerase ... • Attaches to and unzips the DNA molecule to become two separate strands. • Binds to promoter segments of DNA that indicate the beginning of the single strand of DNA to be copied. • Moves along the DNA and matches the DNA nucleotides with a complementary RNA nucleotide to create a new RNA molecule that is patterned after the DNA. The copying of the DNA continues until the RNA polymerase reaches a termination signal, which is a specific set of nucleotides that mark the end of the gene to be copied and also signals the disconnecting of the DNA with the newly minted RNA. The three types of RNA are ... • mRNA (messenger RNA) is transcribed from DNA and carries the genetic information from the DNA to be translated into amino acids. • tRNA (transfer RNA) "interprets" the three-letter codons of the nucleic acids to the one-letter amino acid word • rRNA (ribosomal RNA) is the most abundant type of RNA, and along with associated proteins compose the ribosomes. When the RNA polymerase is finished copying a particular segment of DNA, the DNA reconfigures into the original double-helix structure. The newly created mRNA moves out of the nucleus and into the cytoplasm. Translation is the conversion of information contained in a sequence of mRNA nucleotides into a sequence of amino acids that bond together to create a protein. The mRNA moves to the ribosomes and is "read" by tRNA, which analyzes sections of three adjoining nucleotide sequences, called codons, on the mRNA and brings the corresponding amino acid for assembly into the growing polypeptide chain After the proteins are made, they are packaged and transported to their final destination in an interesting pathway that can be described in three steps involving three organelles: 1. Vesicles transport the proteins from the ribosomes to the Golgi apparatus, a.k.a Golgi complex, where they are packaged into new vesicles. 2. The vesicles migrate to the membrane and release their protein to the outside of the cell. 3. Lysosomes digest and recycle the waste materials for reuse by the cell. Enzymes within the Golgi apparatus modify the proteins and enclose them in a new vesicle that buds from the surface of the Golgi apparatus. The Golgi apparatus is often seen as the packaging and distribution center of the cell.

Discuss how ER proteins are recycled from the Golgi apparatus (2 Steps).

Signaling Mechanism: 1) When vesicles leave from ER to GA, the C-terminus of proteins to be sent back contain a KDEL sequence (tag): Lys-Asp-Glu-Leu-COOH (vesicles contain molecules being transported & other half of ER tag). 2) Golgi has KDEL receptor protein. When vesicles joins cis face, transported molecules are received & tagged proteins return to ER on membrane of empty vesicle.

Compare different views of endoplasmic reticulum provided by thin sectioning, freeze-fracture, and fluorescent microscopy.

Thin Sectioning: ER=light, Mitochondria=Dark. a. Difference in ER visuals between various viewings depend on where section splits. b. When the RER is split at a 90° angle to the surface you see a series of folded lines with visible lumen. Freeze-Fracturing: Can see surfaces exposed; Deep sketch gives deeper view of ER, showing ribosomes sticking to outside of membrane for RER or of smooth wall on SER. Fluorescent Microscopy: Get a "lace-like" network of ER. Can use certain dyes to light up ER

Describe the different morphological types of endoplasmic reticulum (ER).

Three Major Morphological Types of ER: 1. Cisternal Type (Rough ER) a. Resembles flattened sacs b. Ribosomes on surface, producing proteins for export or to be retained in membrane. c. Ribosomes constantly being bound/released from membrane. d. Continuous with outer layer of nuclear envelope e. Membrane-bound vesicles shuttle proteins between RER and Golgi. - Works with Golgi to send new proteins to proper target. 2. Vesicular Type (can be Smooth ER) a. Look like vesicles 3. Tubular Type (can be Smooth ER) a. Ribosomes present in cytoplasm but not on surface of ER = smooth appearance. b. These ribosomes make proteins retained in cell. c. Connected to nuclear envelope d. Smooth ER = Synthesis of lipids, steroids; Metabolism of carbohydrates and steroids; Regulation of calcium concentrations; Drug detoxification.