Cellular Processes: Protein Synthesis, Transport, and Genetics

Posted on Jan 5, 2025 in Biology

Patricia Charlebois-Page – Lecture 1

Topics: ATP, pH, Temperature/pKa, Protein Secretion, Golgi Complex Theories, Protein Synthesis, Protein Export, General Summary of Protein Synthesis

  • ATP provides energy through phosphate binding and conformational changes. Factors affecting ATP hydrolysis include positive binding, ATP and ADP concentrations, pH, temperature, and pKa (lower pH leads to more protonated phosphate).
  • Protein Secretion: Two types of proteins: cytosolic and ER-bound.
  • Signal sequence (S.S.) directs ribosomes to the ER. Protein goes through the ER membrane via a protein channel. S.S. is cleaved. Protein modification (phosphorylation or glycosylation). Some proteins stay in the ER, others go to the Golgi (to organelles or out of the cell). Soluble ER proteins return via COPI (KDEL). Translocons in the ER membrane. Structural complementarity (lock/key complex + receptor on ER membrane). pH, concentration, and temperature affect this.
  • Golgi: (cis/medial/trans). Further protein modification. Vesicle membrane fuses with cis membrane. Lumen (space enclosed by a barrier). Two theories: 1) Vesicles from cis to medial to trans. 2) Cisternal maturation: cis turns to medial, new cis forms by vesicles (including retrograde transport of Golgi proteins). Soluble proteins out of the cell can enter via endocytosis, then sorted to lysosomes for degradation. Some proteins go from trans to lysosomes. Movement via motor proteins and tubules. In vesicles, proteins must bind something. Lysosomes hydrolyze macromolecules with hydrolytic enzymes (lipases, nucleases). Lysosomal pH of 5, cytoplasmic pH of 7, important for acid hydrolases.
  • Protein Export: Secretion is the movement of proteins from the cytoplasm to the inner membrane, periplasm, and outer membrane (out of the cell). As ribosomes translate, the S.S. (hydrophobic region) emerges first, associating with other hydrophobic regions. SRP binds S.S. Soluble proteins are polar (cannot diffuse through non-polar membranes). mRNA and ribosomes are made in the nucleus and transported out. Translation co-translocation: mRNA > free-floating ribosomes > translation > move to RER > SRP recognizes S.S. > SRP stalls translation by binding > ribosome/complex. SRP has RNA + protein (RNA guides, protein activates). SRP binds receptor, moves SRP + complex to translocon (secretion complex). SRP dissociates. Receptor delivers complex to translocon. Transmembrane proteins may have many membrane-spanning regions (hydrophobic). Inner/outer regions have different characteristics due to conditions.
  • Summary of Protein Synthesis: mRNA may be cytosolic or soluble (secreted or CM). Proteins leave the Golgi via vesicles or as part of the membrane, fusing with the target membrane. ER soluble proteins are returned to the ER by COPI-coated vesicles (these proteins have S.S.-KDEL C-term). pH difference between Golgi (slightly acidic) and ER (neutral) may cause receptors to shift conformation, releasing proteins. COPI comes off to allow binding. Clathrin coats vesicles to help invaginate.

Lecture 2

Topics: Receptor-Mediated Endocytosis, LDL, Lipitor, LDL vs. KDEL, GST Fusion, Gene Expression, PLC + SH2 Domains, Processing Gene Information (Prokaryotes vs. Eukaryotes), lac Operon, Bacteria, Archaea, Eukaryotes

  • Endocytosis and membrane fusion are not the same (one remains part of the membrane, the other does not). Cholesterol does not circulate freely; it is packaged in LDL (low-density lipoprotein). Proteins and phospholipids surround cholesterol. This is recognized by LDL receptors on the surface of cells. Adaptor molecules (adaptin) bind the tail of the LDL receptor in the cell. Adaptin recruits clathrin, which coats the membrane. This causes the membrane to bend/invaginate. Vesicles bud off, containing LDL receptors and LDL particles. Vesicles fuse with endosomes (intercellular compartments). Endosomes have a low pH inside. LDL releases its cargo. LDL receptors are recycled to the plasma membrane in vesicles from endosomes. The whole process happens every 10 minutes. LDL particles are delivered to lysosomes to disassemble LDL. Lysosomal hydrolytic enzymes digest the particles, liberating cholesterol and small amino acids. Cholesterol diffuses into the cytoplasm for synthesis. Endosomes have proton pumps. Endosomes can turn into lysosomes by binding to vesicles that carry hydrolytic enzymes. KDEL is a cargo molecule. COP II moves towards the cell membrane; COP I towards the nucleus (retrograde).
  • LDL (low-density lipoprotein): Protein is apolipoprotein B-100 (a large protein). Purposes: i) Create a polar environment and make it soluble (not membrane-bound but contains phospholipids). Non-polar molecules would clump together in the bloodstream. Polar molecules are single individuals in the system. ii) Protein helps attach LDL to LDL receptors. Cholesterol molecules are esterified long-chain fatty acids, surrounded by a lipid layer of phospholipids and unesterified cholesterol. Inside LDL are triglycerides and cholesterol esters. Need cholesterol for: i) Enhancing the permeability barrier properties of the lipid bilayer. ii) Areas with cholesterol are more rigid and less permeable to small H2O molecules at 37 degrees. iii) Preventing membranes from crystallizing at low temperatures. Nervous membranes need cholesterol, as do steroid hormones. Get cholesterol from de novo synthesis and diet. Cholesterol esters are processed mainly in liver or muscle cells. These fats are degraded for energy. VLDL (very low-density lipoprotein) -> LDL (loses triglycerides removed by lipoprotein lipase). Isoenzymes (same function, different tissues). LDL has more protein to fat content. Most fat is from cholesterol. The core of LDL has polyunsaturated fat (lots of double bonds). LDL binds to proteoglycans (large, carbohydrate core + protein) in the blood. Proteoglycans are associated with cartilage hydration and blood membranes. Oxidized LDL attaches with better efficiency. LDL + clumps + proteoglycans and other stuff on the cell membrane = plaque. Attracts macrophages (first cells attracted to pathogen invasion). When plaque gets to small blood vessels, it clogs, leading to stroke or atherosclerosis (plaque breaks at a certain size).
  • Lipitor: (drug prescribed for high cholesterol). Bad cholesterol = LDL. HDL is not really better. The drug interferes with/inhibits de novo (new) synthesis and inhibits salvage (diet) cholesterol. Inhibits the rate-limiting step in the pathway called HMG-CoA reductase. Acetyl CoA (used in fatty acid synthesis). Lipitor also inhibits this. Lower concentration of cholesterol in the blood = more LDL receptors in the liver. Do this to get more cholesterol in the cell. Overall decrease in cholesterol in the blood = less plaque. Some cells prefer salvage over de novo synthesis.
  • LDL vs. KDEL: KDEL receptors cycle between the Golgi and ER. COPI coats, like clathrin, for retrograde transport. Receptors have a high affinity for KDEL in the Golgi due to low concentration. Must have low affinity at the ER due to high concentration. This happens due to pH differences. High affinity = low KM.
  • GST Fusion: GST is a protein that makes mRNA and protein. It gets rid of peroxide. Attaching GST to another protein = long fusion protein. Plasmid (used to hold DNA of interest, a vehicle). Put in a cell to be replicated with the cell’s machinery. Cut DNA with restriction enzymes. Process called ligation > use DNA ligase to cut the vector containing the DNA fragment we cut. Recombinant plasmid. Attach the fragment so it’s not overlapping; it has to be in frame. Put engineered DNA into host cells (E. coli). Process called transformation. Need a promoter of the host cell in the engineered fragment to express the plasmid in the cell. MCS (multiple cloning site). To clone, use a series of unique restriction enzymes. Repressor > ptac > GST > protein fused > stop codons. ptac (2 promoters combined: trp + lac). NcoI has ATG. The vector is man-made. Not all promoters produce the same amount. Need a repressor to control expression. Origin of replication (or2) needs to be replicated. Selectable marker (AMP resistance), gene bla (β-lactamase) degrades AMP protein. ORF in frame with GST protein. Thrombin (recognizes a sequence of amino acids and cuts – protease). GST removes peroxides like radicals from cells (mainly the liver). Affinity chromatography (isolate part of interest). GST binds to glutathione (high affinity – attached to beads). Lyse cells to break them (pass lysate over column). Use thrombin to digest. PLC (phospholipase C – cuts lipase). C = Ca2+. Ca + cAMP = 2nd messengers. PLA + cAMP. Two ways to isolate proteins: by oxidizing glutathione or thrombin. BL21 = E. coli host cells; plasmid inserted into these AMP-sensitive cells.
  • Gene Expression: Have a promoter that initiates transcription. DNA sequence bound by transcription factors (recognize promoter). Transcription starts after the promoter. RBS (ribosome binding site) binds mRNA, not DNA. This is upstream from the open reading frame (ORF). Information for protein is here. Promoter > transcription start site > RBS > ORF. Start codon ATG/AUG (not used). RNA polymerase uses the copied strand as a template. Some genes make just RNA; some make protein because RNA can also have a phenotype. Lag = genes turning on. Stationary phase = stop dividing but don’t die. Cell growth = induction.
  • PLC + SH2: PLC has an SH2 domain (part of a protein that has a specific function). Promoters must be specific per species. PDGF receptor for PLC, GAP, etc. SH2 binds the receptor. Binds to phosphorylated tyrosine. SH2 = amino acid sequence that allows them to bind to P-tyrosines.
  • Processing of Genes: Prokaryotes vs. Eukaryotes: Prokaryotes have no nuclear envelope. mRNA associates with ribosomes as mRNA is being formed. Translation begins before transcription ends. Prokaryotic genes are regulated by one promoter. mRNA contains several genes with related functions on the same mRNA (called an operon). This is how they coordinate synthesis. Eukaryotes have a nuclear envelope. mRNA must finish transcription before translation. mRNA is modified before translation: introns removed, exons spliced together, 5′ cap + 3′ poly-A tail. Translation in the cytoplasm. mRNA usually codes for a single protein.
  • Lac Operon: β-galactosidase (cuts galactose) – gene – lac Z gene. Reading graphs: increasing growth, linear consistent, exponential. Increased β-galactosidase expression. Lactose induces this gene (example of induction). Don’t waste energy. ARG turns off (why? Use in salvage pathway, save energy on de novo synthesis – example of repression). lacI = repressor. Inducer changes conformation. Inducer = allolactose (natural inducer). IPTG (lab inducer). lacI is not part of the lac operon (encodes repressor). lacZ (encodes β-galactosidase). lacY = permease. lacA = thiogalactoside transacetylase. Lac permease is a symporter that moves H+ and lactose into the cell.
  • Bacteria, Archaea, Eukaryotes: Bacteria grow in normal conditions (pH about 7, temperature 25-37 degrees, physiological norms, etc.). Bacteria = prokaryotes (e.g., E. coli). Archaea live in extreme environments (high or low pH, salt concentration, temperature, etc.). A lot of biochemical differences from bacteria. Branched phospholipids in Eukaryotes, not branched. Bacteria and Eukaryotes have ester bonds between fatty acids. They don’t have ether (Archaea has this = more stable and less reactive). Archaea is more related to humans than bacteria. They grow in environments close to ancient Earth.


Lecture 3

Topics: Review of Lecture 2, Endocytosis vs. Exocytosis, Phagocytosis, Comparing Archaea, Bacteria, and Eukaryotes, Eukaryotes vs. Prokaryotes, Gram-Negative vs. Gram-Positive Bacteria, Bacteria vs. Antibiotics, Eukaryotes vs. Plants

  • cAMP involved with the lac operon, silica beads (4 bonds), affinity chromatography. Has to be reversible. Want one thing from this. Proteins bind specific ligands. Elution column filled with resin matrix. Our protein is captured by the matrix while the rest falls out. Add lots of ligand competing with the ligand on the matrix. Ligand + protein flow out of the column. Single pass through the column = 1000 or more purification. Example: ligand = GSH, protein = GST. Steps in cloning: vector = plasmid without the gene of interest. Isolate DNA of interest. Then purify and fragment with restriction enzymes. Capable of binding with DNA cut with the same restriction enzyme. The plasmid must have that restriction enzyme site. Add fragment. Ligase forms phosphodiester bonds. Host cell transformation. Cells plated. Trait desired, isolated, and identified. Example: pGEX-KG = vector.
  • Endocytosis vs. Exocytosis: Prokaryotes. Large/polar molecules can’t cross the membrane. Three major types of endocytosis: i) phagocytosis, ii) pinocytosis, iii) receptor-mediated endocytosis (RME) (also membrane fusion). If the material is bacteria/organic matter = phagocytosis. Liquid = pinocytosis. Specific molecules (e.g., LDL) = RME. Exocytosis = reverse of endocytosis. Removes material at the surface. Exocytosis increases cell SA, increases V, increases membrane proteins, and increases receptors. Endocytosis = opposite. Prokaryotes = 1 circular DNA. Eukaryotes = linear DNA. DNA in both attaches to the membrane (nuclear membrane in Eukaryotes). Mitochondria = circular DNA. Eukaryotes and Prokaryotes = flagella (but move differently in both). The base of the flagella is in the cell wall. Prokaryotes = pili (tube extends). Eukaryotes = no pili. Black in the picture = ribosomes.
  • Phagocytosis: Neutrophils = major white blood cells (engulf microbes by phagocytosis). Monocytes differentiate into a few cell types (macrophages). Use long membranes to grab bacteria. Macrophages are likely to make the first contact with invading pathogens. Macrophages have two functions: i) Kill invaders directly (innate immunity) > bacteria pulled in by vesicles = phagosome > lysosomes + phagosome = cleave macromolecules + ROS generation. ii) Adaptive immunity > major histocompatibility complexes (MHC) > antigens (bacterial parts) placed on MHC = membrane proteins = inside lysosomes, outside the cell. In vesicles = outside the cell > exocytosis out of the cell > cells bind antigens. Macrophages = antigen-presenting cells. Similar to LDL.
  • Compare rRNA: 16S = Prokaryotes (also found in chloroplasts and mitochondria). 18S = Eukaryotes (both similar). Gradient = material stops at a density close to its own. rRNA is more folded = more dense. Eukaryotic ribosome subunits (30s & 50-70s), Prokaryotic (40s & 60-80s). Higher = more dense. rRNA is more conserved than ribosomal proteins (used in the scaffold) = more important function (keeps mRNA in position/enzymatic properties).
  • Gram-Negative vs. Gram-Positive Bacteria: -ve = outer membrane containing lipopolysaccharides (LPS) – lipids = non-polar = anchor, phosphosaccharides in ECM, P = polar properties. Inner membrane, thin peptidoglycans. No teichoic acid. More channel proteins on the outer membrane, less selective transport than the inner membrane. Has DAP in cross-links (direct). +ve = no outer membrane. No LPS. Has thick peptidoglycans (more NAM-NAG-NAM). Teichoic acid. Has lysine in cross-links (bridge). More sensitive to penicillin. Eukaryotes and bacteria = phospholipid bilayer membrane. Eukaryotes = no peptidoglycan (PG). Both have cross-linking = increased structural stability. Both have transpeptidases that do cross-linking. Penicillin attacks this = competitive inhibition. Both have different enzymes. NH2 + COOH or NH3 + COO (in DAP).
  • Bacteria: Have plasmids when selected for = antibiotic resistance or pathogenicity (infection genes). Lowers fitness = more energy used on transcription, translation, etc. = slow growth. Have different shapes (determined by cell wall material). Rod = bacilli = E. coli. Coccoid = sphere = streptococcus (chain of), staphylococcus (branched), diplococcus (2). Extrachromosomal material = viruses, pili, plasmids (mobile DNA). Can sometimes become part of the genome. Virulence = how infectious.
  • Antibiotics: Attack specific structures (e.g., cell membrane). Interfere with cell wall synthesis = better. Bacteria and us have phospholipid membranes (might hurt us). Eukaryotes don’t have peptidoglycans = better. LPS releases endotoxins. Penicillin kills good and bad bacteria. Hard to kill fungi because Eukaryotes have the same DNA polymerase, ribosomes, etc. (have to attack differently). Example: folic acid (folates) = methyl donors CH3 to co-enzymes. dUMP (dihydrofolate) = nucleotide > convert > dTMP (tetrahydrofolate) > goes to TTP to DNA. Enzyme TS (thymidylate synthase) converts dUMP > dTMP. DHFR (dihydrofolate reductase) converts back to dUMP. Co-enzymes oxidize/reduce to be reused. Chemotherapeutic site = DHFR or TS. Bacteria have PABA (makes folates) > folates > DHFR > cycle (target PABA). Target differences in -ve vs. +ve. Macrophages recognize LPS and teichoic acid. AMP affects growing cells (cross-links).
  • Archaea: No bacterial peptidoglycan. Have pseudopeptidoglycan. Lysozyme (digests cell wall, breaks bacteria in the lab) is insensitive to bacteria = opposite.
  • Eukaryotes: (Nucleolus) – rRNA genes + ribosome subunits put together. (Chromatid) – DNA + protein. Histones are positive/basic amino acids. (Nuclear envelope) – 2 not different membranes. The outer membrane is in line with the ER membrane. (Mitochondria/chloroplasts) – 2 membranes, different. Outer = porous, inner = more selective. (Centrosome) – microtubule (main purpose = transport vesicles, etc.) organizing center MTOC. (Cytoskeleton) – actin filaments on the outer edge of the inside of the cell. Actin can be rearranged due to stimuli. Microtubules + actin = dimeric polypeptides (alpha + beta tubulin/actin). PLANT (vacuole) – storage, waste, H2O regulation, salt regulation, cellulose, cell wall, chloroplast > thylakoid membrane = photosynthesis (found in photosynthetic bacteria = thylakoids), no actin but microtubules.
  • Heterochromatin – tightly wound, protein = lots, highly condensed, no/little expression. Euchromatin – less wound, less condensed, gene expression.