INTERCELLULAR TRANSPORT - Crude protein transport: Direct transfer of substances from the cytoplasm to where they are needed. Transport through the membrane: Transfer from source organelles to target organelles. Proteins enter organelles by three mechanisms: - Passes from the cytoplasm into the nucleus through the nuclear pore. - Pass from the cytoplasm into membrane-bound organelles by proteins translocated across the cell membrane. - Transport from the endoplasmic reticulum to organelles through the membrane bubble. The signal sequence is a necessary and sufficient condition for the protein to reach and function in the organelle: the positioning sequences are usually located at the N-terminus (the membrane does not need a signal sequence). Transport mechanism 1: Proteins are transported through nuclear pores - Proteins are transported through the fully folded nuclear pore, the nuclear membrane with the ER membrane continuous. - The nuclear pore complex is a molecular selective gate: Consists of 30 different types of proteins, allowing small hydrophilic molecules to pass through, macromolecules that need a signal sequence to pass through. *How can cells know which amino acid sequence after being translated, which sequence will enter the ER, nucleus,...? It is found that in each aa chain there is an aa sequence, those sequences are like signal chains, this chain enters the ER, another signal chain will enter the nucleus => Cells transport signal protein chains based on aa chain. *How can scientists know which aa chain goes where? People know what sequence each aa chain will have, then fluorescently label it (mark by editing the original DNA to attach additional fluorescent chains) => see where the light spot moves from => thanks microscope (fluorescent substance attached to protein). *It is known that protein A enters the nucleus, protein B enters the ER...what's next? Ideas, predictions => Experimental design => cutting protein chains that are not in the ER (glued to the protein) => seeing it in the ER or cutting ER protein chains. - Transport mechanism: + The R protein has a nuclear localization sequence that pairs with the receptor + When reaching the nuclear pore, the receptor interacts with proteins around the nuclear pore. Due to interactions between receptors and proteins around the nuclear pore. => Destroys interactions between proteins around the nuclear pore, allowing the protein-receptor complex to pass through the nuclear pore (retained by the filament). + In the cytosol, R protein binds to GDP, forming a Ran-GDP complex, which then enters the nucleus. + In the nucleus there is the enzyme Ran GEF => removes GDP (empty receptor) + GTP is abundant in the nucleus => GTP occupies Ran. + High Ran-GTP in the nucleus tends to go out => Ran-GAP removes Pi => GTP becomes GDP. + At this time, the receptor will continue to search to pair with the R protein and begin a new transport process. (nuclear transport receptor is active so that Ran can attach) Transport mechanism 2: Proteins are transported across membranes - The membrane is a rather narrow structure, much smaller than the nuclear pore, where the proteins transported through are very small => Therefore, proteins transported directly across the membrane must be unfolded proteins (and because they are not folded so it only has one amino acid chain pushed in). - Proteins are transported across the nuclear membrane by nuclear transport receptors (protein translocators). - For the ER, there is a process of both transport and translation (co-translational transport). - There are two processes taking place in Cytosol + Process 1: there are free ribosomes suspended in the cytoplasm that capture free mRNA and translate it to create an amino acid chain, then the amino acid chain will be transported across the membrane (a process that usually occurs in the brain). body, chloroplast, etc.) + The second process: the process takes place in the rough endoplasmic reticulum. Ribosomes are attached to the endoplasmic reticulum membrane. When creating an amino acid chain, they will push it into the ER, called the co-translational transport process. ● Process 1: Free ribosomes in cytosol ● Process 2: Ribosomes attach to the membrane of the ER First, ribosomes are small discrete subunits. When there is mRNA -> Ribosomes capture the mRNA chain and begin to translate -> Once it has captured the mRNA chain, the amino acid chain begins to emerge from the Ribosome. There appears an ER localization signal chain -> There is a protein called signal recognition particle (also known as protein recognition) that captures this signal chain -> the complex gate is attached to the SRP receptor -> the corresponding receptor interacts with the translocator protein -> Ribosomes continue to translate and this chain is pushed into the ER. (this is also the formation of a rough endoplasmic reticulum). * When the amino acid chain is pushed into the ER, during the pushing process, the signal chain will often be cut off to avoid returning to the ER due to going somewhere else to work (the protein that cuts off this signal peptide is called signal peptidase). , this signal peptide signal chain will be retained on the ER membrane and through membrane transport will be brought to Lysosomes for decomposition and reuse. - The amino acid sequence determines the active site of the protein: For transmembrane proteins, after being transported inside, there will be a region called the hydrophobic stop-transfer sequence. The signal sequence to know when translation is complete is hydrophobic amino acids. The direction of NH2 and COOH will determine whether the protein functions inside the membrane or outside the membrane -> very important. * Will NH2 always rotate outward, or inward, and what determines NH2's rotation? Hydrophobicity, R radical (phobic or philic), acidic or basic, negative or positive charge, membrane potential will determine its direction. * Proteins that cross the membrane 7 times, how many signal sequences do they have? The attached ribosome continues to translate, requiring 4 protein hydrophobic start-transfer sequences and 3 hydrophobic stop-transfer sequences. - After the amino acid chain moves into the ER, it undergoes folding *What is the folding process, how does it actually happen?Hydrophilicity or hydrophobicity, ion to ion (negatively and positively charged molecules), R radical with OH end => creating covalent bonds, acidic or basic => the most basic of folding . Folding is a process of interaction between amino acids in the amino acid chain. If there is a correct interaction -> correct folding, a large protein will be created. If there is an incorrect interaction -> incorrect folding, it will precipitate. . *Amino acid number 1 is positively charged, numbers 5, 9, 10 are positively charged, so amino acid number 1 will bond with which amino acid number? - Chaperon: Chaperon will support proteins that have not been folded and will have suitable amino acid regions for the chaperon to attach to, leaving only a few parts exposed, then those parts will fold first. When folding the roll sequentially like that, the amino acids will fold correctly => Support the folding. When a protein folds incorrectly, the chaperone will attach because there are empty areas, the cell recognizes it, transports that protein and degrades it. - Not all proteins are completely folded in the ER because when moving through the Golgi, they will continue to fold. There is a signal chain called ER retention signal that helps that chain be retained in the ER and is required, otherwise it will pass through the Golgi, so when ER retention signal this chain will be removed after folding is completed. , to no longer be kept in the ER. *When chaperones attach to amino acid regions, can those regions fold?No, because it is interacting with the chaperon. - There are 2 processes to change the structure of a protein, label and identify that protein: ● Formation of disulfide at cysteine: heating proteins with heat can break weak chemical bonds, but disulfide bonds cannot be broken => using enzymes. When these disulfide bridges are formed incorrectly => Wrong protein => Cannot be repaired => Cells eliminate this protein ● Next to the chaperone, mark with sugar residues and amino acids exposed on the protein surface => see if the protein has finished folding and where it needs to be transported. *How are the road bases marked? When folding a protein, the amino acid chains are transferred sequentially, with an asparagine chain appearing that will be attached to a sugar base when there are 3 amino acids (next is Serine or Threonine). - - Add dyes that will react with a certain chemical radical in a specific protein => dye the protein (dyeing to mark RNA, DNA) => know the radicals of interest in biology. There is an enzyme oligosaccharyltransferase: the saccharide chain (has 14 sugar molecules) and is transferred from lipid molecules to proteins. In the case of mitochondria or chloroplasts, the amino acid chain has been completely translated but it must still be straightened for transport into the mitochondria and chloroplasts by a mechanism similar to that of the ER. - *How are these amino acid chains straightened? Chaperones keep amino acids straight. Each different chaperon will bind to different sequences. Transport mechanism 3: Transport by membrane bubble - The protein is partially folded, then transported to the Golgi (From ER -> Golgi). Golgi is a body with many layers folded together seamlessly. Golgi has two parts, cis and trans, people can distinguish based on the direction of Golgi towards the ER or the cell membrane. Go from ER to Golgi => Transport by membrane bubble. The nature of the membrane bubble is a phospholipid double layer. - Golgi has many folds, proteins are located in the Golgi cavity, proteins move inside for a long time and consume a lot of energy => Transport hops between layers of the Golgi. It is then transported to the cell membrane by the membrane bubble. - Endosome => located intracellularly, divided into 2 types: early because it is located near the cell membrane, late is located closer to the Golgi. - The nature of the membrane bubble is a phospholipid double layer. Budding process: first forms a tumor, then forms a membranous ball, if using an electron microscope => observe a membranous ball like a golf ball. *How does the membrane bubble formation process take place? - There are 3 groups of proteins that support the budding process. - - + There are 3 groups of proteins that support the budding process. Film gloss coating => supports the budding process. COPI and COPII, Clathrin goes from place to place. COPI transports from the Golgi and back to the ER, COPII vice versa. Clathrin is transported from the cell membrane back into the Golgi. Clathrin protein: will be formed by 3 heavy chains and 3 light chains => A protein formed by 6 amino acid chains. The clathrin protein is formed by two genes, one that translates the heavy amino acid chain, and one the light. Clathrin proteins hook together, push up => the sphere protrudes => forms budding. + Mechanism of formation: There is a selection of proteins for transport; for accurate transport, a receptor is required to receive the protein. - Cargo receptors on the membrane move freely to receive cargo molecules => Attract adaptins (intermediate proteins), then the adaptins will attract clathrin => support the budding process. - Once a sphere has been formed, the membrane is pushed up and still attached, requiring dynamin, which is responsible for cutting off the remaining membrane area, forming a clathrin-coated sphere. - After forming the coating, adaptin and clathrin coating are on the outside and will be removed from the membrane bulb => the bare membrane bulb is transported to the germinal organelle. *Where do the proteins located on the membrane of the membrane appear? The proteins located on the membrane bulb originate from the donor organelle. + Mechanism of fusion with source organelles (membrane fusion): *Why does membrane fusion occur? Which properties? When hydrophobic molecules are close together, their contact with water is minimal => then there is a membrane fusion process and this process comes from hydrophobic interactions. *Where does the signal peptide that is transported to the Golgi membrane go? What are the characteristics of proteins on the ER membrane?Enters the ER, then transported through the Golgi via the membrane bubble. Exocytosis and endocytosis: - Exocytosis: brings lipid molecules, proteins and carbohydrates to the cell surface. - Endocytosis: Taking fluids and molecules from the outside into the cell. *How do we know how the cell attaches the uplink? Each different protein, after folding, will reveal different amino acid segments => Sequence recognition => Sugar base attachment. + + + + + + + Proteins are further modified inGolgi apparatus Proteins enter the Golgi via vesicle fusion with the cis face and passageGolgi between successive compartments by means of transport vesicles. Oligosaccharide chains added to the ER are modified by enzymes in the Golgi -Add and remove sugars to create complex oligosaccharides. The reaction is progressive - the enzyme acts early in the cis compartment, acts late in the cis compartmentThe enzyme operates in the trans compartment. Exocytosis occurs through two distinct mechanisms road: Continuous exocytosis: A steady flow of delivery occurs in all cells. Plasma Membrane components to replace intracellular materials and for membranes evolution. XRegulatory melanocytes: Only active in cells specialized for secretion (ie secretory cells cells in the intestines and glands). Secretory vesicles are attached to the plasma membrane until the cell receives an external signal. There are two main mechanisms of endocytosis: Phagocytosis: “cell eating”; Ingestion of large particles (i.e. microorganisms,cellular debris) through large vesicles called phagosomes. Occurs only in specialized cells. Pinocytosis: “cell drinking”; The swallowing of liquids and small molecules through the small passageway (<150 nm diameter) vesicles. Occurs in every cell. - Receptor-mediated endocytosis is a specialized form of pinocytosis + + + Pinocytosis randomly traps molecules in the extracellular fluid. Receptor-mediated endocytosis traps specific molecules, concentrating them blisters. Both processes utilize clathrin-mediated vesicle formation.
