Molecular Cell Biology Bulk transport: Receptor mediated endocytosis

Transcription

1 Paper : 15 Module :07 Development Team Principal Investigator : Prof. Neeta Sehgal Department of Zoology, University of Delhi Co-Principal Investigator : Prof. D.K. Singh Department of Zoology, University of Delhi Paper Coordinator Content Writer Content Reviewer : Prof. Kuldeep K. Sharma Department of Zoology, University of Jammu : Dr. Poonam Sharma, Gargi College, University of Delhi Dr. Jasvinder Kaur, Gargi College, University of Delhi Ms. Poornima Vishwakarma, Research Scholar, DU : Prof. Rup Lal Department of Zoology, University of Delhi 1

2 Description of Module Subject Name Paper Name Module Name/Title Module ID Keywords Zool 015: Bulk transport M07: Receptor mediated endocytosis Receptor-mediated endocytosis, Clathrin, pits, Adaptor proteins, Accessory proteins, Dynamin, Cholesterol, Transferrin, Transcytosis Contents 1. Learning Outcomes 2. Introduction 3. Types of coated vesicles in vesicular trafficking 4. Clathrin coated vesicles and pits 4.1. Adaptors and accessory proteins 4.2. Coat Formation, Maturation and Invagination 4.3. Components of the endocytic pathway 5. Receptor-mediated endocytosis 5.1. The LDL Receptor and endocytosis of cholesterol 5.2. Endocytosis of Transferrin bound iron 5.3. Few endocytosed proteins remain within the cell 5.4. Transcytosis 6. Summary 2

3 1. Learning Outcomes By the end of this module, you should be able to learn receptor-mediated endocytosis, particularly clathrin-mediated endocytosis. The various adaptor and accessory proteins involved will be briefed. You will also be acquainted with the mechanisms involved in the uptake of cholesterol and iron by cells. 2. Introduction Endocytosis is an energy using process by which biomolecules (mainly large polar) are transported inside a cell by engulfing them (Fig. 1). Endocytosis can be subdivided into four types: Receptor-mediated endocytosis (Fig. 3), caveolae, pinocytosis and phagocytosis (Fig.2). Fig. 1: Endocytosis involves engulfing of biomolecules by the cell membrane. A vesicle is formed by the invagination of plasma membrane and is pinched off, taking along the cargo inside. Source: o0e8bjvz1gy/t6_eyx_kovi/aaaaaaaaali/rjwn9o2dvum/s1600/endocytosis.jpg Clathrin-mediated endocytosis is mediated by the formation of small vesicles that are coated with the cytosolic protein clathrin. Clathrin-coated pits are found on plasma membranes of all cell types. Caveolae are flask shaped pits (special type of lipid raft) in the plasma membrane of some cell types and resemble the shape of a cave (hence the name caveolae). They consist of caveolin- a cholesterol binding protein. Most endocytosis using caveolae, 3

4 deliver contents of vesicles to lysosomes or other organelles. However, when material endocytosed using caveolae is released into the cytosol, it is termed Potocytosis.These are especially abundant in endothelial cells, and adipocytes. These are rich in proteins and lipids such as cholesterol and sphingolipids and have several functions in signal transduction. They also play a role in mechanosensation, endocytosis, and the uptake of pathogenic bacteria and certain viruses. Pinocytosis occurs from highly perturbed regions of the plasma membrane. Extracellular fluid and material within it is engulfed within the invagination of the plasma membrane. These invaginations containing the fluid are then pinched off inside the cell. This a nonspecific process (Video 1). Phagocytosis involves the uptake of larger membrane areas. In this process, cells bind and engulf larger particulate matter such as dust particles, cell debris, apoptotic cells and microorganisms (Video 1). Fig. 2: Endocytosis. In phagocytosis, cells bind and engulf larger particulate matter. The non-specific process involving fluid uptake along with the material is pinocytosis. While, receptor-mediated endocytosis is specific involving coated vesicle formation. Source: 4

5 Video 1: Types of endocytosis: Phagocytosis, Pinocytosis and Receptor-mediated endocytosis. Source: There are few other mechanisms through which ligands can be brought into the cell. However, clathrin-mediated endocytosis is the best studied mechanism. Receptor-mediated endocytosis or clathrin-mediated endocytosis is one of the most significant processes with which viruses and bioparticles can enter or leave eukaryotic cells. It is an endocytic mechanism by which selective molecules are ingested or absorbed into the cell. Receptor-ligand interaction imparts specificity to the process.receptors are present on the plasma membrane of the target tissue, which specifically bind to the ligands on the outside of the cell. These ligands can be hormones, metabolites, proteins or even viruses in certain cases. The inward budding of plasma membrane vesicles then result in endocytosis. Fig. 3: Receptor-mediated endocytosis is an endocytic mechanism by which selective molecules are ingested or absorbed into the cell. These ligands can be hormones, metabolites, proteins. Source: C AEF9E0.png 5

6 3. Types of coated vesicles in vesicular trafficking Three types of coated vesicles can be formed, each type specific for a particular route, transporting proteins from certain parent organelle to certain destination/target organelle. All of these vesicles are formed by reversible polymerization of a distinct set of protein subunits. Clathrin coat mediates transfer of vesicles that bud from the trans-golgi network and the plasma membrane and that then fuse with late endosomes. Vesicles with a COP II coat, transport proteins from the rough ER to the Golgi.Finally,COP I facilitates retrograde (backward) transport from the trans-to the medial-to the cis-golgi (between golgi compartments), as well as from the cis-golgi to the rough ER (where they were originally synthesized). Here COP refers to the specific coat protein that initiates the budding off from the cis-golgi (Fig. 4). Fig.4a: COP I is involved in retrograde transport of vesicles, back to the ER where they were synthesized or between golgi compartments. COP II traffics vesicles in the forward direction; whereas clathrin is involved in endocytosis from plasma membrane and transports vesicles budded off from trans-golgi. Source: ea75/s377book3chapter12_f016hi.jpg 6

7 Fig.4b: Vesicular trafficking: Adaptor proteins are associated with clathrin-mediated transport. AP-2 helps with endocytosis from plasma membrane; AP-3 from trans-golgi to lysosomes and AP-1from trans-golgi to endosomes. Source: 4. Clathrin coated vesicles and pits Clathrin-coated pits have been implicated in many intracellular transports of molecules. It plays an important role in hormone receptor signaling. This is done either by delivering the bound receptor-ligand to intracellular compartments for optimum signaling or by removing the bound receptor and weakening the signal. The molecules that are transported may include transferrin, LDL-cholesterol and growth factors such as epidermal growth factors. The process begins when such a molecule binds on the cell surface. This is followed by assembly of coat components to form clathrin-coated pits at the under surface of the plasma membrane. The internalized receptors then interact with molecules called adaptors and become clustered into the growing pit. The vesicles containing the receptor-ligand complex, accessory and regulatory proteins, are then pinched off and the vesicles uncoated. The receptors and ligands are then sorted through intracellular compartments Adaptors and accessory proteins Adaptors represent a diverse group of proteins recognizing different classes of cargo receptors. Adaptor proteins AP1, AP2, AP3 and AP4 are the best characterized classes of adaptor proteins (APs). Each of these four classes is localized to different intracellular 7

8 compartments. All adaptor proteins differ in their cargo specificities and are constructed on fundamentally similar plan. Each adaptor protein consists of two kda large subunits (a beta-class adaptin, and one of either an alpha, gamma, delta or epsilon adaptin), a 50 kda medium chain mu adaptin and a kda small chain sigma adaptin. Upon formation of the receptor-ligand complex on the plasma membrane, adaptor proteins bind to the plasma membrane. AP2 adaptor interacts with the plasma membrane via. binding sites for PIP2 (Phosphatidylinositol-4,5-bisphosphate; Fig. 5). Whereas, for membrane binding, the other APs, require GTP bound form of the G protein. The cytoplasmic portion of the receptors in turn contains sequence motifs that are recognized by adaptor proteins. A variety of accessory proteins help coordinate the coat assembly. Accessory proteins such as CALM (Clathrin-assembly lymphoid myeloid leukaemia protein), promote clathrin and AP2 binding. Fig. 5: Receptor-ligand complex formation is mediated by the adaptor proteins. This interaction is stabilized by clathrin. Source: %20Lics%20e%20Lics%20com%20Mests/MD/1%C2%BAANO/1%C2%BASEM/11- UBA3/Biologia%20Celular/UBA3%20T%20BC%20transp%20endomembranar%20e%20sintese%20prot%20( C)/VER%20complementos/Receptor%20Mediated%20Endocytosis_files/REMRecogSequence1.jpg 4.2. Coat Formation, Maturation and Invagination 1. The PIP2 binding site helps the adaptor AP2 to attach to the under-surface of the plasma membrane (Fig. 5). 2. This interaction is stabilized by clathrin (unassembled). The unassembled cytosolic form of clathrin forms a triskelion shape comprising of three heavy and three light chains (Fig.6a and b). 8

9 3. Clathrin also activates phosphorylation of mu-2 subunit. The phosphorylated mu-2 subunit (green) swings out from its normal position in the AP2 core and bind receptors. 4. Clathrin then assembles onto the adaptors to form the outer layer of the coated vesicles. The triskelia assemble to form a polyhedral lattice like structure that surrounds the vesicle (clatratusin latin, a lattice; Fig.6c). 5. The triskelia act as a mold in which the vesicle grows. 6. Clathrin stabilizes the curvature until the growing pit invaginates to form a closed vesicle. These closed vesicles have prominent pentagonal and hexagonal faces (Fig.6c). 7. In the final stages of vesicle formation,pip2 s signal transduction is inhibited. This is due to dephosphorylation of PIP2 by phosphatases such as synaptojanin 1 (Synj1). Synj1 has been shown to be recruited by endophilin 1 which possesses a curve-sensing role (Fig. 9). 8. As a final part of this invagination and budding off, protein amphiphysindimerises onto the neck of the membrane and stabilizes it. The GTPase enzyme dynamin binds to amphiphysin and derives membrane cleavage. These are actin-binding proteins belonging to the BAR (Bin/Amphiphysin/Rvs) superfamily (Fig. 8). Fig.6a: Structure of clathrin and the clathrin-coated vesicle: A. Clathrin is a triskelion shaped protein with three heavy and three light chains B. Clathrin assembles to form a coat around which vesicle develops. Source: 9

10 Fig.6b: Anatomy of clathrin-coated vesicle. Source: Fig.6c: Electron micrograph of clathrintriskelia (unassembled; below) and polymerized clathrin where pit grows. (Source: 9. Dynamin acts as a sensor for the maturation of the CCP (Clathrin-coated pits) to a CCV (Clathrin-coated vesicle). In the presence of GTP, dynamin will undergo a twisting action that results in its breakage (Fig. 7). 10. Dynamin binds to auxilin and ATPase Hsp70. These act together to uncoatclathrin following abortion of the vesicle from the plasma membrane. 10

11 11. Hsc70 proteins are powered by the hydrolysis of ATP. Three ATP molecules are required for the disassembly of a single clathrintriskelion from the cage. 12. Uncoating reaction is sensitive to ph.chaperones are recruited and bind at ph6.0 and Hsc70 mediated dissociation of the clathrintriskelion occurs at ph7.0 (Fig. 9). 13. Uncoating of APs and accessory proteins is thought to be brought about separately by synaptojanin. 14. Microtubule network traffic is involved in the sorting of CCVs to early endosomes in mammals. Fig. 7: Dynamin acts as a sensor for the maturation of the CCP to a CCV. In the presence of GTP, dynamin will undergo a twisting action that results in its breakage. Source: Fig. 8: Formation of clathrin-coated pits and their invagination. The black outline on the coated vesicle represents the location of an individual clathrintriskelion. Source: 11

12 Fig. 9: After coat assembly, bud/pit formation starts till the vesicle is budded off by dynamin. In the final stages of vesicle formation, Synj1 has been shown to be recruited by endophilin 1 which possesses a curve-sensing role. Clathrin and other accessory proteins are uncoated and the vesicle goes on to fuse with early endosome. Source: Components of the endocytic pathway In mammalian cells the endocytic pathway comprises of separate membrane sections. 1. Most vesicles coming from the cell surface encounter the first compartment of the endocytic pathway, the early endosomes. These are often located in the periphery of the cell. They have a characteristic tubulo-vesicular structure and a mildly acidic ph. In the acidic ph of the compartment, the endocytosed ligands dissociate from their receptors. The receptors are then recycledthrough tubules to the cell surface. 2. Late endosomes represent the second compartment of endocytic pathway. They are acidic (approx. ph 5.5)and often contain lysosomal membrane glycoproteins and acid hydrolases. These are thought to mediate a final set of sorting events prior to delivery of material to lysosomes. 3. The last compartment of the endocytic pathway is lysosomes. The approximate ph of a lysosome is 4.8. Their major function is to break down cellular waste products and other macromolecules into simple compounds. These are then returned to the cytoplasm as new cell-building materials. 12

13 5. Receptor-mediated endocytosis In receptor-mediated endocytosis, a specific receptor on the cell surface recognizes and then binds a ligand (extracellular macromolecule); the plasma-membrane region containing this receptor-ligand complex is then endocytosed, becoming a transport vesicle. The rate at which a ligand is internalized is limited by the amount of its corresponding receptor on the cell surface. Most common ligands internalized by receptor-mediated endocytosis are cholesterolcontaining particles called low-density lipoprotein (LDL); transferrin, an iron-binding protein; insulin and most protein hormones etc The LDL Receptor and endocytosis of cholesterol Cholesterol is insoluble in body fluids and thus transported through a water-soluble carrier. Low-density lipoprotein (LDL) is one such carrier. Its outer surface consists of a single layer of phospholipids and cholesterol, in which the protein apo-b is,embedded (Video 2). The nonpolar inside core is of cholesterol. The cholesterol is esterified to a longchain fatty acid, mainly linoleic acid through the single hydroxyl group. After endocytosis, the LDL particles are transported to lysosomes. Apo-B protein is degraded to amino acids, whilst cholesterol esters are simplified to cholesterol and fatty acids by lysosomal hydrolases. The cholesterol is incorporated directly into cell membranes or is re-esterified and stored as lipid droplets in the cell. Cholesterol also is converted to steroid hormones in adrenal cortical cells and to bile acids in hepatocytes. Analysis of mutant LDL receptors has revealed that a four-residue sequence in the cytosolic domain is crucial for internalization: Tyr-X-X-ø, where X can be any amino acid and ø is a bulky hydrophobic amino acid, such as Phe, Leu, or Met. A mutation in either tyrosine or ø residues abolishes the ability of the receptor to be incorporated into clathrin-coated pits. Endocytosed cell-surface receptors dissociate from their ligands within late endosomes.beginning 15 minutes after internalization, ligands are transferred to lysosomes, but the intact receptors themselves usually are not found in these organelles. Instead, the receptor-rich elongated membrane vesicles that bud from the late endosomes mediate the recycling of receptors back to the cell surface. The spherical part of the late endosome eventually with their cargo of ligand, soon fuse with lysosomes (Fig. 10). 13

14 The insulin and LDL receptors, bind their ligands tightly at neutral ph but release their ligands if the ph is lowered to 5.0 or below. The late endosome is the first vesicle encountered by receptor-ligand complexes with a ph this low and hence is the organelle in which these and most other receptors dissociate from their tightly bound ligands (Video 3). Clathrin-coated vesicles, endosomes and lysosomes contain a V-class ATP-dependent proton pump and a Cl channel. This allows a significant H+ concentration gradient to be generated. Most cell-surface receptors that undergo endocytosis will repeatedly deposit their ligands within the cell and then recycle to the plasma membrane, once again to mediate the internalization of ligand molecules. For instance, the LDL receptor has a 20-hour life span in which it makes one round trip into and out of the cell every minutes. In contrast, the receptors for insulin and other growth factors, after binding the ligand generally cycle only two or three times before being degraded in the lysosome - reducing the number of cellsurface receptors and thus the sensitivity of the cells to hormone signaling. Video 2: Receptor-mediated endocytosis of cholesterol containing LDL particle. Source: 14

15 Video 3: The late endosome is the first vesicle encountered by receptor-ligand complexes with a low ph and hence is the organelle in which LDL receptors dissociate from their tightly bound ligands. Cholesterol is simplified in lysosomes and the receptors are recycled. Source: Fig. 10: Fate of an LDL particle and its receptor after endocytosis. Ligands are transferred to lysosomes, but the intact receptors are recycled back to the surface via. tubular portion of late endosomes. The spherical part of the late endosome eventually with their cargo of ligand, soon fuse with lysosomes, where the ligand is cleaved to simpler forms. Source: 15

16 5.2. Endocytosis of Transferrin bound iron Unlike the LDL pathway, the receptor-ligand complexinvolving the transferrin receptor and its liganddoes not dissociate in late endosomes. However, in this case also, changes in ph mediate the sorting of receptors and ligands. Transferrin transports iron to all tissue cells from the liver and intestine. Apotransferrin, ironfree form, binds two Fe3+ ions very tightly to form ferrotransferrin. All growing cells contain surface transferrin receptors that bind ferrotransferrin at neutral ph, receptor-bound ferrotransferrin is then endocytosed. The two Fe3+ atoms remain in the cell. The apotransferrin part of the ligand however, is secreted from the cell within minutes, carried in the bloodstream to the liver or intestine, and reloaded with iron. The transferrin receptor ligand complex shows a unique ability. The apotransferrin remains bound to the transferrin receptor at the low ph ( ) of late endosomes. At a ph of less than 6.0, ferrotransferrin dissociates to form two Fe3+ atoms which are transported from the late endosome vesicle into the cytosol (Fig. 11). The apotransferrin thus formed remains bound to the transferrin receptor and is recycled back to the surface along with the receptor. Remarkably, although apotransferrin can remain bound to its receptor at a low ph of 5.0 or 6.0, it does not bind at neutral ph. Upon fusion of the recycling vesicles with the plasma membrane, the bound apotransferrin dissociates from its receptor. This is due to the neutral ph of the extracellular interstitial fluid. The surface receptor is then free to bind another molecule of ferrotransferrin. 16

17 Fig. 11: Following endocytosis, in the acidic late endosome compartment, iron is released from the receptorferrotransferrin complex. The apotransferrin protein however, remains bound to its receptor at this ph, and they recycle to the cell surface together where the neutral ph of the exterior medium causes release of the apotransferrin to be loaded with iron again. Source: Few endocytosed proteins remain within the cell Coated pits were first discovered in insect eggs, where they occupy a large portion of the plasma membrane. In some receptor-ligand systems, such as in oocytes (egg cells), endocytosed material simply remains in the cells without processing. This material in a hen s egg is several grams of protein, nearly all of which is imported from the bloodstream by receptor-mediated endocytosis. Vitellogenin is one such example. These yolk proteins remain in storage form within the egg and used as a source of amino acids and energy by the developing embryo. Egg-white proteins such as ovalbumin and lysozyme are endocytosed by the egg cell. 17

18 5.4. Transcytosis This process of transcellular transport combines endocytosis and exocytosis. It is employed to import an extracellular ligand from one side of a cell, carry it across the cytoplasm, and secrete it at the opposite side of the plasma membrane. Movement of maternal antibodiesacross the intestinal epithelial cells of the newborn follows the process of transcytosis. This owes to the differences in ph of the extracellular media on the opposite sides of intestinal epithelial cells in newborn. This allows unidirectional movement of immunoglobulins from the lumen to the blood. 6. Summary Receptor-mediated endocytosis or clathrin-mediated endocytosis is one of the most significant processes with which viruses and bioparticles can enter or leave eukaryotic cells. It is an endocytic mechanism by which selective molecules are ingested or absorbed into the cell. Receptor-ligand interaction imparts specificity to the process.the molecules that are transported may include transferrin, LDL-cholesterol and growth factors such as epidermal growth factors. The process begins when such a molecule binds on the cell surface. Upon formation of the receptor-ligand complex on the plasma membrane, adaptor proteins bind to the plasma membrane. AP2 adaptor interacts with the plasma membrane via. binding sites for PIP2. The PIP2 binding site helps the adaptor AP2 to attach to the under-surface of the plasma membrane. This interaction is stabilized by unassembled clathrin unassembled (video 4). The unassembled cytosolic form of clathrin forms a triskelion shape comprising of three heavy and three light chains.this is followed by assembly of coat components to form clathrin-coated pits at the under surface of the plasma membrane. The triskelia act as a mold in which the vesicle grows. The internalized receptors then interact with adaptors and become clustered into the growing pit. Clathrin stabilizes the curvature until the growing pit invaginates to form a closed vesicle. These closed vesicles have prominent pentagonal and hexagonal faces.the vesicles containing the receptor-ligand complex, accessory and regulatory proteins, are then pinched off and the vesicles uncoated (Fig. 12). The receptors and ligands are then sorted through intracellular compartments. 18

19 Fig. 12: The receptor-ligand complex is bound to adaptors and is stabilized by unassembled clathrin molecules. Coated pit formation continues till the curve sensing protein dynamin acts to break off the pit to form CCV. Clathrin and other proteins are now disassembled and the transport vesicle containing the receptor-ligand complex is now ready to fuse with an early endosome. Source: icking.resources/2f98be78afa53ee41e d08d2.jpeg Most common ligands internalized by receptor-mediated endocytosis are cholesterolcontaining particles called low-density lipoprotein (LDL); transferrin, an iron-binding protein; insulin and most protein hormones etc.low-density lipoprotein (LDL) is a carrier for cholesterol, which is transported from liver and intestine to the rest of the body tissues.after endocytosis, the LDL particles are transported to lysosomes. Apo-B protein is degraded to amino acids, whilst cholesterol esters are simplified to cholesterol and fatty acids by lysosomal hydrolases. The cholesterol is incorporated directly into cell membranes or is reesterified and stored as lipid droplets in the cell. Cholesterol also is converted to steroid hormones in adrenal cortical cells and to bile acids in hepatocytes.ldl receptors, bind their ligands tightly at neutral ph but release their ligands if the ph is lowered to 5.0 or below. The LDL receptors dissociate from the ligands in the late endosomes and the tubular portion of the late endosomes enriched with receptors then fuses with the plasma membrane to be recycled and reused. Unlike the LDL pathway, the receptor-ligand complex involving the transferrin receptor and its ligand (iron) does not dissociate in late endosomes. At a ph of less than 6.0, ferrotransferrin dissociates to form two Fe3+ atoms which are transported from the late endosome vesicle into the cytosol (and remain there). The apotransferrin (iron-free form) thus 19

20 formed remains bound to the transferrin receptor and is recycled back to the surface along with the receptor. At the neutral ph of the extracellular interstitial fluid, apotransferrin dissociates from its receptor and recycled back to be reused. In some receptor-ligand systems, such as in oocytes (egg cells), endocytosed material simply remains in the cells without processing (Vitellogenin). On the other hand, movement of maternal antibodies across the intestinal epithelial cells of the newborn follows the process of transcytosis. Video 4: Overview of clathrin-mediated endocytosis. Source: 20