SCBM 251 Molecular Trafficking Assoc. Prof. Rutaiwan Tohtong Department of Biochemistry Faculty of Science rutaiwan.toh@mahidol.ac.th
Lecture outline 1. What is molecular trafficking? Why is it important? 2. The key mechanisms of molecular trafficking 3. The Signal Hypothesis : The discovery, The mechanism, The importance 4. Vesicular Transport: What is it? How is it important? How it happens? 5. Transport into the nucleus & nuclear localization signal 6. Transport from exterior to interior of the cell
Eucaryotic cells contain membrane-bound organelles to segregate and to organize the different chemical reactions/metabolic processes What will happen if we do not keep each compartment/organelle separated? Figure 12-1 Molecular Biology of the Cell ( Garland Science 2008) Figure 12-2 Molecular Biology of the Cell ( Garland Science 2008)
Communication is needed within the cells, between the organelles, as well as between the interior and exterior of the cells. Some occur through free (unregulated) diffusion, others by regulated processes. Figure 13-3b Molecular Biology of the Cell ( Garland Science 2008) Figure 12-6 Molecular Biology of the Cell ( Garland Science 2008)
Secretion of digestive enzymes from pancreatic acinar cells into the digestive tract can be studied by the pulse-chase experiment After a 3 min pulse with radioactive aa, autoradiography revelaed that newly synthesized proteins were localized to rough ER. Following a chase with nonradioactive aa, proteins were found to move from the ER to the Golgi, then to secretory vesicles, to the plasma membrane and cell exterior, respectively Fig 11.2 The Cell: A Molecular Approach, 7 th Ed.
Mechanisms of Molecular Trafficking Three main mechanisms by which membrane-bound organelles import proteins Targeting of some proteins to a specific destination/organelle requires a specific transport mechanism and/or a signal sequence Table 12-3 Molecular Biology of the Cell ( Garland Science 2008)
1. Transport Across Membranes Figure 12-36c Molecular Biology of the Cell ( Garland Science 2008) Figure 12-36a Molecular Biology of the Cell ( Garland Science 2008) ER has a central role in lipid & protein biosynthesis. The ER membrane is the site of production of all the transmembrane proteins & lipids for most of the cells organelles including ER, golgi, lysosomes, endosomes, secretory vesicles, plasma membrane ER membrane also makes most of the lipids for mitochondrial & peroxisomal membranes Almost all proteins to be secreted or those destined for the lumen of ER, golgi or lysosomes, are initially delivered into the ER lumen Intracellular Ca2+ storage
Transport Across Membranes Figure 12-38 Molecular Biology of the Cell ( Garland Science 2008) Figure 12-6 Molecular Biology of the Cell ( Garland Science 2008)
Nobel Prize in Physiology or Medicine 1999 for the discovery that proteins have intrinsic signals that govern their transport and localization in the cell www.nobelprize.org Günter Blobel 1936-2018
How are ribosomes engaged in the synthesis of secreted proteins directed to the ER membrane? Ribosomes engaged in the synthesis of secreted proteins attach to membranes, where as those engaged in the synthesis of cytosolic proteins do not (Cesar Milstein) Ig light chain produced by in vitro translation contain N-terminal 20 aa that are not present in the secreted light chains (Blobel & Dobberstein) The Signal Hypothesis: these N-terminal 20 aa direct the binding of the ribosomes to the ER membrane during translation of proteins to be secreted
Blobel G & Dobberstein B, 1975 J Cell Biol. 1-In vitro translation of Ig light-chain mrna on free ribosomes S-Secreted Ig light-chain 2- Ig light-chain synthesized by in vitro translation on membrane-bound ribosomes 3-Products of in vitro translation on membrane-bound ribosomes were resistant to protease digestion, suggesting that they were protected from proteases by insertion into microsomes
Understanding how secreted proteins are transferred into the ER provides the framework of understanding of how the proteins are targeted to all the other membrane-enclosed compartments of the cell, thereby impacting virtually all areas of cell biology 1. As the signal sequence emerges from the ribosome, it is recognized and bound by the signal recognition particle (SRP) 2. The SRP escorts the complex to the ER membrane, where it binds to the SRP receptor 3. The SRP is released, the ribosome binds to the translocon, and the signal sequence is inserted into the membrane channel 4. Translation resumes, and the growing polypeptide chain is translocated across the membrane 5. Cleavage of the signal sequence by signal peptidase releases the polypeptide into the lumen of the ER
Signal sequences direct proteins to the correct compartment Signal peptide Signal patch Proteins destined for the ER possess a 12-60 aminoterminal signal sequence which is necessary & sufficient to direct them to the ER, where as those destined to remain in the cytosol lack this sequence Alberts Alberts Panel 12-1
2. Transport by Vesicles (Vesicular Transport) During vesicular transport, membranous carrier structures bud off a donor compartment and fuse with a recipient one, thus delivering their membrane-associated as well as its luminal content to the target organelle. Figure 13-3 Molecular Biology of the Cell ( Garland Science 2008)
Vesicular transport mediates transport of the biosynthetic-secretory pathways (outward) as well as endocytic pathways (inward) Transport vesicles bud off from one compartment and fuse with another while carrying material from the lumen of the donor compartment as cargo Figure 13-1 Molecular Biology of the Cell ( Garland Science 2008) Figure 13-2 Molecular Biology of the Cell ( Garland Science 2008)
Nobel Prize in Physiology or Medicine 2013 for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells" James Rothman Randy Schekman Thomas Südhof www.nobelprize.org
How would you show that transport between the golgi cisternae is mediated by vesicular transport?
Three types of coated vesicles distinguished by their coat proteins Proteins to be transported within cells contain structural information that guides them to their correct destinations. Figure 13-4 Molecular Biology of the Cell ( Garland Science 2008) Figure 13-5 Molecular Biology of the Cell ( Garland Science 2008)
Clathrin-coated vesicles COPII-coated vesicles Figure 13-8 Molecular Biology of the Cell ( Garland Science 2008) Figure 13-7c, d Molecular Biology of the Cell ( Garland Science 2008) Figure 13-20 Molecular Biology of the Cell ( Garland Science 2008)
The cytoskeleton guide, and motor proteins propel, the vesicles/organelles to the right destination Once a vesicle has budded off, it does not move passively but rather is guided toward the target membrane by the filamentous structures of the cytoskeleton. Vesicles move along cytoskeletal tracks, either microtubules or actin filament, and with the help of motor proteins such as kinesin and dynein, hydrolyzing ATP to propel movement of vesicles or organelles along these filaments and organize directional membrane flow.
How is the specificity in vesicular transport regulated? The vesicle is tethered and docked near the target membrane and subsequently fuses with the acceptor bilayer, releasing Its contents into the target/acceptor compartment. This crucial process depends on two types of proteins: Rab proteins direct the vesicle to specific spots on the correct target membrane, and SNARE proteins mediate the fusion of the lipid bilayers Figure 13-14 Molecular Biology of the Cell ( Garland Science 2008)
The SNARE proteins catalyze the membrane fusion in vesicular transport. Membrane fusion is important in other processes besides vesicular transport. Fusion of plasma membranes of sperm & egg during fertilization, myoblast fusion during development of multinucleated muscle fibers. All cell membrane fusions require special proteins and are tightly regulated to Ensure that only appropriate membrane fuse. Entry of virus including HIV virus requires membrane fusion that may use similar mechanism to SNARE-mediated fusion. Figure 13-16 Molecular Biology of the Cell ( Garland Science 2008) Figure 13-19a-b Molecular Biology of the Cell ( Garland Science 2008)
Transport from Golgi to Lysosomes Golgi Processing/Sorting glycoproteins from ER Attach carbohydrates/oligosaccharides to proteins/lipids that are sent from ER Synthesis of glycolipids & sphingomyelin Lysosomes A major site of intracellular digestion Contain 40 hydrolytic enzymes: proteases, nucleases, glycosidases, lipases,phospholipases, phosphatases, sulfatases All are acid hydrolases (ph 4.5-5) Figure 13-3b Molecular Biology of the Cell ( Garland Science 2008)
Transport from Golgi to Lysosomes Mannose 6-phosphate (M6P) are added to N-linked oligosaccharides of the lysosomal enzymes in the cis Golgi. The M6P serves as the marker for targeting to the lysosome. Figure 13-43, 13-44 Molecular Biology of the Cell ( Garland Science 2008)
I-Cell Disease (Mucolipidosis type II) GlcNAc phosphotransferase deficiency Lysosomal enzymes do not acquire M6P in cis Golgi Almost all hydrolytic enzymes are missing from the lysosome of fibroblasts, but instead are found in blood because they fail to sort properly in the Golgi Hydrolases are secreted rather than transported to lysosomes Undigested substrates accumulate in lysosomes, forming large inclusions in patient s cells
3. Transport through the nuclear pores The nuclear pore complexes control the traffic of molecules between the nucleus and the cytoplasm, separated by a double lipid bilayer Transport through nuclear pore complex is gated or selective, although some small molecules pass freely through the nuclear pore complex by passive diffusion. However, macromolecules (proteins & RNAs) are recognized by specific signals and selectively transported through the nulear pore complexes Figure 12-8, 12-9 a-c, 12-10 Molecular Biology of the Cell ( Garland Science 2008)
Transport through the nuclear pores Alan Smith et al. (1984) Figure 12-16b Molecular Biology of the Cell ( Garland Science 2008) Proteins with nuclear localization signal are recognized by importins, which work in conjunction with Ran, a GTPbinding protein, to transport proteins through the nuclear pore complex How would you show that the nuclear localization signal is necessary and sufficient to target proteins for into the nucleus? Figure 12-11 Molecular Biology of the Cell ( Garland Science 2008)
Transport into the cell from the plasma membrane: Endocytosis Page 787 Molecular Biology of the Cell ( Garland Science 2008)
Receptor-mediated endocytosis The LDL receptor dissociates from its ligand LDL, in the early endosome and is recycled back to the plasma membrane for reuse, leaving the discharged LDL to be carried to lysosomes. Influenza and HIV viruses also gain entry into cells through receptor-mediated endocytosis Figure 13-53 Molecular Biology of the Cell ( Garland Science 2008)
GM. Cooper et al. The Cell: A Molecular Approach 7 th ed. chapters 11 and 12 B. Alberts et al. Molecular biology of the Cell 5 th ed. chapters 12 and 13