Membrane Trafficking: Secretory and Endocytic Pathways PDF

“Membrane trafficking”: Secretory and Endocytic Pathways Learning Objectives: Understand the destinations of proteins synthesized on the ER Understand the mechanisms of vesicular transport between membrane-bound compartments (“membrane trafficking”)...

“Membrane trafficking”: Secretory and Endocytic Pathways Learning Objectives: Understand the destinations of proteins synthesized on the ER Understand the mechanisms of vesicular transport between membrane-bound compartments (“membrane trafficking”) Understand the roles of coat proteins, adaptors, SNAREs, tethers, and Rab proteins Understand function of the Golgi Understand principles of receptor mediated endocytosis (uptake of LDL) Understand how large particles are taken up by phagocytosis  How do proteins move from the ER to the plasma membrane, the Golgi, endosomes, and lysosomes? Golgi Apparatus Endosome and Lysosome Plasma membrane and secreted proteins oadmap of vesicle transport (“membrane trafficking”  Nuclear envelope rough ER smooth ER Transpor t vesicles Golgi Transpor t vesicles 1µm Figure 13-25b Molecular Biology of the Cell (© Garland Science 2008)  Vesicle transport: budding and fusion The asymmetry of the lipid bilayer is e.g. ER preserved, as is the orientation of membrane proteins e.g. Golgi transport is selective: only certain specific proteins are selectively incorporated into transport vesicles Figure 13-2 Molecular Biology of the Cell esicle transport maintains membrane topolog Explain “topology” Endoplasmic Reticulum Golgi Apparatus Cytosol Plasma Membrane outside The lumen of the ER, the lumen of transport vesicles, and the lumen of the Golgi correspond topologically to the ‘outside’ of the cell The regions of proteins with N-linked glycosylation are on the lumenal side and therefore topologically on the ‘outside’ of the cell Figure 13-2 Molecular Biology of the Cell Today’s topics 1. Overview of membrane trafficking pathways 2. Mechanisms of vesicle budding, movement, tethering and fusion 3. The Golgi apparatus 4. Endocytosis 5. Lysosomes and autophagy e transport pathways (“membrane trafficking”) f all eukaryotic genes have an ER signal sequence Nucleus ER ‘resident’ protein Protein to be secreted out of cell Selective Protein to be delivered to the lysosome transport occurs by vesicles e transport pathways (“membrane trafficking”) f all eukaryotic genes have an ER signal sequence Nucleus ER ‘resident’ protein Protein to be secreted out of cell Selective transport of Protein to be delivered to the lysosome protein “cargos” occurs by vesicles Today’s topics 1. Overview of membrane trafficking pathways 2. Mechanisms of vesicle budding, movement, tethering and fusion 3. The Golgi apparatus 4. Endocytosis 5. Lysosomes and autophagy  The typical transport vesicle “life-cycle”: budding, movement, tethering, and fusion Note: the term “cargo” can refer to any protein that is moved from one place to another by a vesicle Reinisch, Ferro-Novick, Developmental Cell 2007  Vesicle budding: cargo selection and membrane shaping 1. Selection of lumenal cargo to be transported is mediated by cargo receptors Coated Vesicle COPII Coat Transpor Adaptors t Vesicle Cargo recepto r Cytosol ER Membrane ER lumenal Lumen cargo Modified from: Figure 15-21 Essential Cell Biology  Vesicle budding: cargo selection and membrane shaping 1. Selection of lumenal cargo to be transported is mediated by cargo receptors 2. Cargo adaptors capture transmembrane protein cargos (including cargo receptors) by binding “sorting signals” in their cytoplasmic tails Coated Vesicle COPII Coat Transport Cargo adaptor Vesicle (“adaptins”) Cargo recepto r Cytosol ER Membrane ER lumenal Lumen cargo Modified from: Figure 15-21 Essential Cell Biology  Vesicle budding: cargo selection and membrane shaping 1. Selection of lumenal cargo to be transported is mediated by cargo receptors 2. Cargo adaptors capture transmembrane protein cargos (including cargo receptors) by binding “sorting signals” in their cytoplasmic tails 3. Coat proteins bind adaptors to shape membrane into a vesicle Coated Vesicle Coat Vesicle Transpor Cargo adaptor t Vesicle (“adaptins”) Cargo recepto r Cytosol ER Membrane ER lumenal Lumen cargo Modified from: Figure 15-21 Essential Cell Biology  Vesicle budding: cargo selection and membrane shaping 1. Selection of lumenal cargo to be transported is mediated by cargo receptors 2. Cargo adaptors capture transmembrane protein cargos (including cargo receptors) by binding “sorting signals” in their cytoplasmic tails 3. Coat proteins bind adaptors to shape membrane into a vesicle 4. Vesicle pinches from membrane Coated Vesicle Vesicle Coat Transpor Cargo adaptor t Vesicle (“adaptins”) Cargo recepto r Cytosol ER Membrane ER lumenal Lumen cargo Modified from: Figure 15-21 Essential Cell Biology  Vesicle budding: cargo selection and membrane shaping 1. Selection of lumenal cargo to be transported is mediated by cargo receptors 2. Cargo adaptors capture transmembrane protein Note: we often cargos (including cargo receptors) by binding refer to both the “sorting signals” in their cytoplasmic tails ‘adaptor 3. Coat proteins bind adaptors to shape membrane proteins’ and the into a vesicle ‘coat’proteins 4. Vesicle pinches from membrane together as “the 5. Vesicle is uncoated and ready to fuse with target coat” of a vesicle membrane Coated Vesicle Vesicle Coat Transpor Adaptors t Vesicle Cargo recepto r Cytosol ER Membrane ER lumenal Lumen cargo Modified from: Figure 15-21 Essential Cell Biology  “Coated” Vesicle Transport Pathways: coats and adaptors Coats and their associated cargo adaptors define the AP-2 / protein composition of Clathrin transport vesicles: cargo receptors are ‘recycled’ COPII AP-1 / to be reused Clathrin COPI -> recycles cargo receptors to the ER Each cargostands “COP”: adaptor recognizes for ‘coat protein’ different sorting signals. COPI Many cargos have more than one sorting signal – to reach their final destination Type of Coated Vesicle Origin Destination COPII Endoplasmic Reticulum cis-Golgi COPI cis-Golgi Endoplasmic Reticulum Clathrin / Adaptin-1 Trans-Golgi Network Early Endosome (“AP-1”) Clathrin / Adaptin-2 Plasma Membrane Early endosome More on clathrin later this lecture! (“AP-2”)  Movement: many vesicles (and organelles) are moved along microtubules by molecular motors Transport vesicle rganelles and vesicles are connected to motors by “Rab” GTP-binding p These Rab GTPases are also important for tethering…  Tethering: Rab GTPases recruit tethering proteins Molecular motor Rab-GEF GDP Rab-GTP GTP Rab-GDP Microtubule Rab-GAP Modified from Figure 15-22 Essential Cell Biology  Fusion: “SNAREs” drive the fusion of transport vesicles with their target membrane motor v-SNARE = vesicle SNARE t-SNARE = target SNARE Rab-GTP Microtubule Rab-GAP Modified from Figure 15-22 Essential Cell Biology  SNARES play a central and essential role in membrane fusion Requires ATP-hydrolysis “Tethering”“zipper formation” SNARE bundle disassemb led After fusion and SNARE bundle disassembly, v-SNAREs are recycled back to donor compartment in different vesicles (i.e. COPI vesicles return the COPII vesicle v-SNARE back to the Figure 15-23 ER) Essential Cell Biology  Mutated membrane trafficking proteins disease Yarwood et al. 2020 (do not memorize) Today’s topics 1. Overview of membrane trafficking pathways 2. Mechanisms of vesicle budding, movement, tethering and fusion 3. The Golgi apparatus 4. Endocytosis 5. Lysosomes and autophagy  The Golgi apparatus (aka Golgi complex) is a stack of flattened, membrane-enclosed sacs called cisterna Vesicles from the endoplasmic reticulum Functions of the Golgi Apparatus: 1. Elaboration of the N- linked oligosaccharide chains “Glycosylation” 2. Sorting to multiple destinations in the trans Golgi Network (‘TGN’): Endosomes plasma Endosome and Lysosomes membrane lysosome Plasma membrane Figure 15-26 Essential Cell Biology  Secretory components are sorted in the TGN for the constitutive and regulated exocytic pathways (“secretory granule”) Figure 15-30 Essential Cell Biology The TGN sorts traffic to multiple destinations Protein synthesis on rough endoplasmic reticulum. Protein folding and N- linked glycosylation Modification, sorting Nucleus and transport out of the Golgi complex Constitutive secretion Transport, delivery and fusion at Regul appropriate ated s ecre tion destination Selective transport to the Golgi complex by COPII trans Golgi network vesicles cis medial trans ‘TGN’ Today’s topics 1. Overview of membrane trafficking pathways 2. Mechanisms of vesicle budding, movement, tethering and fusion 3. The Golgi apparatus 4. Endocytosis 5. Lysosomes and autophagy  Endocytosis Endocytic recycling Nucleus Constitutive secretion Regul ated secre tion Endocytosis: The taking in (‘uptake’) of material by the invagination of the plasma Modified membrane. from Fig. 15-18 Essential Cell Biology 3/e  The “LDL” receptor on the cell surface binds LDL and internalizes it through clathrin coated pits “LDL” = Low Density Lipoprotein is a “particle” made of proteins and lipids, and is used by your body to transport fat, lipids, and cholesterol through your bloodstream Clathrin - coated pit (with AP-2 cargo adaptor)  Clathrin coated pits See ECB movie 15.5 from inside the cell looking at the inner surface of the plasma membran Figure 15-19b Essential Cell Biology ake is an example of “Receptor-Mediated Endocytosis See ECB Movie 15.12 (also posted on Canvas) early Low pH (about 6.0) If the endosome is neutralized to pH 7, both the LDL and receptor get trapped there and the receptor does not recycle. Many other proteins have specific receptors and are taken up by ‘RME’. ‘RME’ is also used to remove membrane proteins from the surface, when no longer needed, one example is the Figure 15-33 Epidermal Essential Cell BiologyGrowth Factor (EGF) Receptor that we will talk (see movie 15.10)  The lumen of lysosomes is acidic and full of hydrolytic enzymes The lumen is acidic due to the presence of a ‘proton’ (hydrogen ion) pump. Lysosomes contain hydrolytic enzymes that can break down any biological molecule. Lysosomal enzymes function optimally at pH 5.0. They are collectively called ‘acid hydrolases’. Lysosomes contain membrane transporters so that products of degradation can be reused by the cell. Fig. 15-35 Essential Cell Biology 3/e  LDL Receptor LDL Receptor recycling Nucleus Constitutive secretion Regul ated secre tion Notice: Coats and adaptors not shown Modified from Fig. 15-18 Essential Cell Biology 3/e  Endocytosis is exploited by many viruses to gain entry into cells Virus particle binds to cell surface receptor For example: Flu virus HIV Polio virus Hepatitis C virus Coronavirus  Sign the attendance sheet, if PollEV did not work for you today