Cell sorting, endocytosis and exocytosis 17-10-23.docx

Proteins are transported into organelles by 3 mechanisms. All synthesis begins in the cytosol except mitochondrial and chloroplast proteins that are synthesised on ribosomes inside the organelles. Amino acid sequence can contain a sorting signal that directs the protein to the organelle in which it...

Proteins are transported into organelles by 3 mechanisms. All synthesis begins in the cytosol except mitochondrial and chloroplast proteins that are synthesised on ribosomes inside the organelles. Amino acid sequence can contain a sorting signal that directs the protein to the organelle in which its required. Different sorting signal directs proteins into different organelles. Proteins that lack such sorting signals stays in the cytosol. The protein must be transported across an organelles membrane and there are 3 mechanisms to move the protein as its impermeable to hydrophilic macromolecules. Gated transport Proteins moving from cytosol to nucleus are transported via nuclear pores. Doesn’t cross the membrane. Guided by signal sequences by the help of the soluble receptor protein in the cytosol that guides it across the nuclear pore. Bidirectional movement Nucleoporins forms nuclear pores. Smaller molecules can diffuse freely but active transport is needed for molecules like DNA and RNA polymerases. Transport across membrane Proteins transported by protein translocators located in the membrane. The transporter protein must unfold for the translocator to guide it across the hydrophobic interior of membrane. For protein moving from cytosol to mitochondria and ER. Rough ER Coated with ribosomes. Captures proteins from cytosol during synthesis. Soluble proteins and transmembrane proteins are types of proteins transferred from cytosol to ER Soluble proteins- made in ER Fully transport through membrane Ends up in lumen of ER. Signal peptidases cleaves of the signal sequence on the protein. N-terminal sequence initiates translocation. Transmembrane proteins Partly transferred across membrane Remains embedded in membrane. Goes through protein translocator. N-terminal sequence initates translocation Signal sequence is cleaved off by signal peptidases. The hydrophobic amino acids act as stop transfer sequence forms an a-helix transmembrane protein and stays in the bilayer. The stop transfer sequence is never removed from the polypeptide. Transmembrane proteins that span the membrane more than once must go through the process above several times. Transport into the ER is co-translational for a soluble protein. As the protein is being translated, N-terminal signal sequence is made first and recognised by signal recognition particle (SRP) which then then recognised by SRP receptor embedded on ER membrane. This brings ribosomes down to ER membrane and gives its rough appearance. SRP is then released, and the protein passes through protein translocation channel. Summary of how soluble and transmembrane proteins passes through the ER membrane. The protein which is bound to ribosome contains a signal sequence made up of hydrophobic amino acids which is guided to the ER membrane by signal recognition particle which binds to signal sequence. Protein synthesis is slowed down until the SRP binds to SRP receptor bound to the ER membrane. SRP is released and ribosomes passes through the protein translocator channel. Signal sequence also has a function of opening the channel by which the peptide chain threads through. The signal peptide remains bounds to the channel. Signal peptidase then cleaves off the signal sequence and released into the membrane from the channel and degraded. With the transmembrane proteins N-terminal signal sequence initiates the translocation. It contains stop-transfer sequence as well as signal sequence. The signal sequence is eventually cleaved off by the signal peptidase, but the stop-transfer sequence remains embedded in the membrane. The process can happen several times if the transmembrane protein spans the membrane more than once. Vesicular transport- requires ATP Each transport vesicle must only take proteins appropriate to its destination. Relies on membrane budding and fusion. Carries soluble proteins. Transmembrane protein stays in the membrane of vesicle, fuses with destination compartment and stays in the membrane. Between ER and Golgi. Exocytosis pathway Step 1- Secretion Co-Translational transmembrane transport into the ER from the ribosome where they are synthesised. Soluble proteins fully translocate into ER lumen whilst transmembrane proteins partially translocate and stay imbedded in the membrane. Step 2- Secretory pathway Vesicular travel of protein from lumen of ER pinching off and fusing with Golgi Step 3- Travels through Golgi and modifies the proteins (glycosylation) Step 4- Exits golgi via secretory vesiscles. Step 5- Exocytosis takes place. Constitutive vs regulated secretion. Regulated secretion is when a signal such as hormone or neurotransmitter causes the vesicle to fuse to the membrane and secrete. Constitutive secretion happens all the time and doesn’t require signal. Carries both transmembrane and soluble proteins. Modifications to proteins during secretion N-linked glycosylation in the ER Chaperones proteins in the ER ensures quality. Hold the proteins in the ER unless proper folding or assembly occurs. O-linked glycosylation in the Golgi Most proteins are covalently modified in the ER. Most proteins that enter the ER lumen are converted into glycoproteins by covalent attachments of short and branched oligosaccharides- glycosylation. Functions of glycosylation includes protection from degradation, function in recognition. Sugars are not added one by one, a preformed branched oligosaccharide containing 14 sugar is attached en bloc to all proteins. Glycosylation is N-linked and happens on asparagine residue. Sugars are transferred from lipid dolichol onto Asn residue of the peptide chain being translocated into the ER. O-linked glycosylation in the Golgi. Sugars binds to OH groups of amino acids side chains. Less frequent than N-Linked Protein coats drive vesicle budding. Vesicles that bud from membranes have a distinctive protein coat on their cytosolic surface. After budding from parent organelle, the vesicle sheds it coat allowing the membrane to directly interact with the membrane it will fuse to. Clathrin and COP (coat proteins) are major components. 2 functions of the coat Helps shape the membrane into a bud. Helps to capture molecules for onward transport. Different compartments have vesicles coated with different coat proteins e.g., clathrin coated vesicles start from Golgi and end up in lysosomes via endosomes. Once a vesicle reaches the target, it must recognise and dock with the specific organelle. Each vesicle displays molecular markers on its surface. These markers are recognised by specific receptors that are complementary to markers on vesicles. All depends on monomeric GTPases called Rab proteins that ensure specificity of vesicular transport. Rab proteins are recognised by tethering proteins on cytosolic surface of target membrane. Additional recognition is provided called SNAREs. Once tethering protein has captured the vesicle by holding onto its Rab proteins, SNAREs on vesicles interact with complementary SNAREs on target membrane and firmly docks the vesicles in place. SNAREs also play a role in catalysing the membrane fusion. Endocytosis PM Early endosomes Late endosomes lysosomes PM Early endosomes recycling endosomes PM 2 types of endocytosis- both required energy Pinocytosis Occurs slowly in fibroblast. Mostly carried out by clathrin coated vesicles. After pinching off, the coat sheds rapidly and fuses with an endosome. Receptor mediated. Destination of endocytosed cargo /////////////////////// //////////// Phagocytosis Pseudopodia forms and extends around the particle to engulf. Tip fuses to form phagosomes. Phagosomes fuse with the lysosomes and digest large particles. E.g., macrophages and neutrophils Lysosomes Contains about 40 types of hydrolytic enzymes and active in acidic conditions. Lysosomes digests endocytosed material.