Introduction Into Cell Membrane Trafficking Flashcards
What did George Palade Discover?
- Secretory Pathway
- 1950s-1960s
- Used EM & membrane fractionation to study protein fraction.
- Golgi was linked to secretion.
Secretory Pathway
(1) ER -> Protein synthesis begins.
(2) Golgi apparatus -> proteins processed and sorted.
(3) Secretory granules -> proteins packaged for secretion.
(4) Cell surface -> proteins are released outside cell.
What were key techniques used by Palade?
- EM and autoradiography (used to trace proteins as move through secretory pathway)
- pulse-chase (first direct evidence, proposed that transport vesicles move proteins between compartments, laid understanding for protein trafficking)
How does pulse-chase experiment work?
(1) Pulse phase:
cells briefly exposed to radioactive tritium-labeled amino acids, became incorporated into newly made proteins.
(2) Chase phase:
radioactive aa removed, allowing researchers to track where labeled proteins move over time.
What is the autoradiography process?
(1) Cells labeled with tritium-tagged amino acids while alive.
(2) Samples fixed, embedded with resin (epon), and sectioned for EM.
(3) Photo-sensitive silver emulsion - placed over sample.
(4) Stored in dark and low temperature to allow radiation to expose to emultion.
(5) Developed into metallic solver grains, visible under EM.
What are two major organelles of secretory pathway?
(1) ER
- newly synthesized proteins inserted.
- folding.
- N-linked glycosylation.
- quality control.
(2) Golgi Apparatus
- N-linked oligosaccharides modified
- sorting to various destinations occurs (cell surface, secretory granules, endosomes)
What are the categories of Protein Transport Between Compartments?
(1) Transmembrane Transport.
- Proteins from cytoplasm -> ER, mitochondria, peroxisomes, chloroplasts.
(2) Gated Transport.
- Proteins move through nuclear pores to go in and out of nucleus.
(3) Vesicular Transport.
- Involved vesicles that transport proteins between organelles.
- Vesicles bud off from one organelle and fuse with another (e.g., between ER and golgi).
(4) Direct Connections.
- ‘kiss and run’ temporary fusion between vesicles & membranes in endocytotic pathway.
- golgi stacks may also have direct connections for transport in the secretory pathway.
(5) Protein Diffusion.
- some proteins detach and diffuse through cytoplasm, only possible for peripheral proteins on membranes outside.
What are the steps of Vesicular Trafficking?
(1) Sorting of cargo, budding, seperation, from source membrane.
(2) Transfer to destination.
(3) Storage (e.g., synaptic vesicle)
(4) Recognition of target membrane fusion.
What happens in the first step of vesicular trafficking (sorting of cargo, budding, seperation, from source membrane.) ?
- Coat proteins -> cytoplasmic proteins (e.g., clathrin) help form vesicles by binding to cytoplasmic domain of cargo proteins.
- coat proteins concentrate cargo in vesicles, vesicles bud off from source membrane.
- once cargo is sorted, coat proteins are lost, vesicles free to move and fuse with target membrane.
- tubular transport intermediates exist during transport process.
What are the different coat proteins?
(1) Clarithrin - first coat discovered and highly visible. Forms vesicles at cell surface, golgi, endosomes (signals, tyrosine, dileucine signals)
- ArfI protein involved clarthin-coated vesicle formation on Golgi and endosomes.
(1) COPI
Found only on Golgi.
Golgi -> ER
Trafficking not on endosomes, recognize dilysine signals.
(2) COPII
- only on ER
- Involved in vesicle formation for exit from ER
- recognize diacidic and diphenylalanine motifs.
What happens in the second step of vesicular trafficking? (Transfer to Destination)
- some vesicles travel long distances within the cell, requiring motor proteins to move along cytoskeleton.
- motor proteins help move vesicles by binding to microtubules or actin filaments.
What are the microtubule motor proteins?
(1) Dynein
- moves towards (-) end of microtubules (cell centre in fibroblast) - moving things towards nucleus.
(2) Kinesin
- Moves toward (+) end of microtubules (cell periphery in fibroblast)
What is the motor protein for actin filaments?
- Myosin
short distance transport
What happens in the last step of vesicular trafficking? (Recognition of target membrane and fusion involves two key steps)
- Tethering proteins ‘docking factors’ help initially recognize the target membrane. Often works with Rab proteins. (RECOGNITION)
- SNARE proteins (v-snare on vesicle and t-snare on target membrane) are responsible for FUSION of vesicle with target membrane.
- SNARE proteins are organelle specific.
