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Topic 3 - Cellular Transport
Most proteins are synthesised in the cytosol, but proteins destined for organelles contain sorting signals,
usually targeting them to specific organelles down the endomembrane system.
The fates of proteins after synthesis is determined depending on intrinsic signals (peptide motifs) within their sequence (eg: KDEL=ER retention), which targets them to specific organelles.
The Endomembrane System [ER-Golgi-CM]
The Endomembrane System is a series of compartments involved in the synthesis and transport of specific lipids, proteins and glycolipids.
The Endoplasmic Reticulum is the entry point of cellular proteins into the endomembrane system, and then non-ER residents are transported via vesicular transport, which buds from the donor compartment and fuses with the acceptor compartments
The flow of the membrane is tightly controlled, balanced, directional and each compartment keeps its distinct identity despite the extensive flow of membrane flowing.
Discovery of the Endomembrane System
James Rothman - The Endomembrane system was identified by a Biochemical approach by James
Rothman, who recapitulated membrane transport within a test tube using VSVG assays.
Randy Shekman - It was also studied with a Genetic approach by Randy Schekman. He used Yeast to investigate mutations in genes and correspond these to defects in membrane transport at non-permissive temperatures (so they would all grow initially). As yeast is such a simple organism, the mutations in budding could be seen under an electron microscope. The relevant mutated genes were then identified
Ways to Study Organelles
Super Resolution Microscopy - Standard light microscopy has a limited resolution which is often greater than the size of many cellular structures. Super Resolution Microscopy uses ways to get around this and produce resolutions down to 20nm, using types such as STORM (Stochastic Optical Reconstruction)
Electron Microscope Tomography - The other methods of microscopy need a thin section to be observed,
but Electron Tomography allows 3D images to be constructed to avoid this. It does this by taking a thick section of the cell, and powerful electron beams are fired to penetrate the sample, and the section is rotated to generate a 3D image.
GFP Technology - GFP is an intrinsically fluorescent protein found in jellyfish, and it can be attached to any protein using DNA technology, allowing it to be tracked in the cell using live cell microscopy.
Transport vesicles are coated and are made up of a characteristic protein coat. These coat proteins act as mechanical devices to induce membrane curvature and select the components for inclusion into the vesicle.
3 Classes of coat protein have been identified:
Clathrin, COPI and COPII.
Clathrin is used by the Plasma Membrane and Trans-Golgi Network. COPI is used to get across the Golgi and return components from the cis-golgi back to the ER. COPII is used by the ER to get to the Golgi.
Clathrin-coated vesicles have a characteristic basket-like structure, made up of 3x heavy chains and 3x light chains in a triskelion structure, which is formed through the self-assembly of Clathrin molecules.
Clathrin requires specific adaptor proteins to assemble into the triskelion structure and these are the 2 nd major component.
AP Complex (Tetrameric Adaptors) - The AP complexes are heterotetramers with 2 large, 1 medium and 1 small subunit (adaptin). The heavy chains bind to Clathrin, and the light chains bind to the cargo, allowing the
Clathrin to become linked to the cargo.
In addition to binding cargo, AP complexes can also be linked to the membrane (eg: AP2 is used for
Endocytosis) through small GTPases and membrane lipids, and these are called Phosphoinositides.
Phosphoinositides, are only found on the cytoplasmic leaflet and are generated through the differential phosphorylation of the Inositol rings.
Their function is to recruit proteins, which have a specific modular binding domain, to the membrane.
They can be hydrolysed to generate second messengers or act as signals in their own right.
PIP2 - In the cytoplasm AP2 is in a closed condition and is unable to interact with its cargo. AP2 binding to
PIP2 causes a conformational change to occur, which now allows it to bind to its cargo. But if there is no PIP2 present than AP2 cannot be recruited.
Dynamin is a GTPase which polymerises into a ring structure around the bud neck and recruits other proteins.
It is used to 'pinch off' the vesicle through GTP hydrolysis, acting as a 'pinchase'.
This function can be proven with Shibire Flies, which caused them to become paralysed, due to their inability of the Clathrin coated vesicles to pinch off at the neuromuscular junctions.
Clathrin-Coated Vesicle Formation Summary 1) PIP2 is present and activates AP2.
2) AP2 binds to the Cargo, allowing the recruitment of Clathrin, binding the Cargo to the Clathrin.
3) Clathrin helps deform the membrane but are also assisted by BAR domains (Amphiphysin), which help the membrane form a bud.
4) Dynamin is then recruited to the neck of the bud and undergoes a conformational change upon GTP
hydrolysis to mediate membrane pinching.
Following budding of the vesicle the coat vesicle coat must be removed, so the coat proteins are dissociated and removed. This is necessary for fusion with the target membrane, and is mediated bby the ATPase hsp70,
that unwinds the Clathrin legs and PIP2 phosphatase, that removes PIP2 from the membrane.
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