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Lecture 3

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University of Toronto St. George
Jane Mitchell

LECTURE 3: INTRACELLULAR COMPARTMENTS AND PRTEIN SORTING Slide 2: intracellular compartments - Percentage of the total volume of the cell that’s occupied by different compartments - Data based on a liver cell - Cytosol occupies the largest volume of the cell o It’s the location where protein synthesis starts, also where degradation protein and some other molecules happen, degradation and sythensis of small moelcules, synthesis of glycolysis. So many things happen in the cytosol which are not taergeted to a special compartment - Mito: o Location where energy is made in the cell  ATPase protein uses proton motive force to make ATP from ADP and phosphate - ER (rough/smooth) – different functions. Depending on the cell, it can have more ER or vice versa - Nucleus: where DNA is transcribed and replicated - And other smaller compartments Slide 3: definition of organelle - Different types of orgnaleles classified according to their appearance - Examples of organelles that are not membrane bound: o Nucleolus:  Specific compartment within the nucleus where ribosomal RNA is transcribed and it has a different appearance than the rest of the nucleus o Centrosomes:  Important role in organizing the cytoskeleton o These were identified using the electron microscope. That is why these are classified along with the other membrane enclosed compartments. Slide 4: polarized cell: - Apical domain which looks different from the basal domain - Epithelial cell of the small intenstine - Apical domain as the vili - Basal domain is flat - Lysosomes: involved in degrading components brought into the cell through endocytosis and also degrading used organelles no longer functioning properly - Endosomes: an early compartment from endocytosis - Peroxisomes: where oxidation reactions occur - You can see the granular appearance of Ribosomes from electron microscope: responsible for protein synthesis  the main func of rough ER - Smooth ER: involved in phospholipid synthesis and detoxification of hydrophobic compounds - Golgi apparatus: located just adjacent to ER  involved in processing protein and adding more glycosylation’s to proteins before they’re delivered to the plasma membrane or other organelles Slide 5: cell viewed under electron microscope - Of a Liver cell - Viewed using an electron microscope - nucleus can see the condensed chromatin in the nucleus and the membrane of the nucleus - All of these membrane components of the internal organelles actually make up a much larger fraction of the lipid component of the cell than the plasma membrane does - This is specially true to the ER, which is a branched set of tubules compacted into a small space Slide 6 - The % of total cell membrane - The plasma membrane makes up quite a SMALL component of the cellular membrane - They have a different proportion of RER to SER. - ER makes a huge component of the cellular membrane - The pancreatic cell has mostly ROUGH ER with very little low smooth ER and it’s the opposite for the liver hepatocyte  main functions is to make digestive enzymes and to secrete them through exocytosis, therefore to cover it with ribosomes that translate mRNA into proteins - Liver hepatocyte has more of the smooth ER and that’s b/c the liver plays an important role in detoxification and the smooth ER is where a lot of the hydrophobic molecules are broken down so they can be excreted from the body  therefore smooth ER makes a big portion of liver cells - There is lots of detoxification in the liver  so when hydrophobic molecules are broken down there should be lots of SER to detoxify the blood stream - There are different amounts of different organelles in different cells. Allows them to perform different functions - Cells can response to their ennnviroemtn and change the amount of organelles in their cells. - Example: In cases where a certain drug is continually present in the blood stream and is continually being detoxified by the liver, liver cells can make additional ER and proteins that degrade those drugs and allow cells to better deal with that amount of drugs in the blood stream Slide 7: intracellular compartments are dynamic and there are temporal connections b/w them - Exchange both lipids and protein components - Some of these components make up an important sys called the endomembrane system and that allows proteins synthesized in the rough ER to be transported from rough ER in vesicles to golgi where they might be heavily glycosylated and then from the golgi in transport vesicles to the plasma membrane In the case of a protein been targeted to the plasma membrane. - Or for a components that needs to be secreted they move from the golgi in the secretory vesicles and fuse with a execretory vesicles and from the process of exoxytosis the compnents will be secreted to the lumen. - These Compartments they don’t have an obvious connection between them eg: one of the small vesicles and the ER of the golgi, but they are connected over time because it helps move both lipid and protein components to be moved from the ER to the golgi and out to the plasma membrane. - The process of endocytosis completes the circle because ecompents of te plams ammebrane can be converted into endosomes through the process of endocytosis, and plasma membrane can be moved into the cell that way. - All of these connections between these organelles, they need to get proteins from their site of synthesis in the RER, ad membrane proeins requires to get them to the plasma membrane, and also it is required to get the SER that needs to be delivered to the plasma membrane as the cell grows - All these are accomplished in a regulate and organized way - This system is called endomembrane system Slide 8: - Different organelles that connects this system is called an endomembrane system - It’s a subset of intracellular compartments temporally connected through vesicles that transport proteins and lipids between them , as well as the luminar components - All function together in a specific pathway - ER, golgi apparatus, lysosomes vacuoles or plants as well as vacuoles. Part of the endomembrane system - Mito and chloroplasts are not considered to be a part of the endomem sys b/c they have a diff function (to produce energy) Slide: biosynthetic/secretory pathways - Way in which these compartments are temporally linked - They function in this biosynthetic or secretory pathway - In the biosynthesis of proteins and lipids made in the ER and delivered to other organelle membranes within the cell or the process of secretion for secreted proteins and for that case the contents move out of the cell through exocytosis - There’s also an endocytic pathway: o Where contents move into the cell through endocytosis o ER is a site where proteins are synthesized  example of a membrane associated protein  assume it’s a Na-K pump. the protein then has to get to the plasma membrane and it does so by moving from rough ER by a transport vesicle and the vesicles fuses with the golgi and golgi is where a lot of glycosylations are added to proteins and then the protein moves through the different systems of the golgi and move through transport vesicles as well and then bud off from golgi and move towards plasma mem and eventually fuse with plasma membrane and that fusion delivers the luminar contents of the vesicle out into the extracellular environment and it also delivers the lipids and proteins to the plasma membrane. Na K pump is now part of the membrane and it reaches its destintions. o There’s also the reverse pathways = the endocytic pathway which contents can be moved into the cell  after a certain amount of time the cell would like to regulate the amount of Na-K pump on the plasma mem and so parts of the plasma mem could be endocytosed and that would transfer any proteins that were in the vasinity of the endocytosis into a vesicle that would be moved into the cell and the contents would be degraded o All of these processes are called cycular transport  transporting components throughout the endocytosis system either out of the cell or into the cell or they’re just transported to one of the cellular compartments where they need to function. For example a protein that needs to be found in the ER wouldn’t be transported out into the plasma membrane Slide 10 - Exocytosis: contents exiting the cell o Delivers vesicle contents into the extracellular space and vesicle space becomes part of the plasma membrane  a way that a plasma membrane protein that needs to function on the plasma membrane needs to get to its final destination - Endocytosis : o Plasma membrane in the end forms the vesicle membrane and the vesical luminal contents come from the extracellular space o Engulfs particles and takes them into the cell o Also a way in which membrane proteins or lipids that are in the plasma membrane can be internalized back into the cell and removed from the plasma membrane o Remember that the plasma membrane and the vesicle is a bilayer with 2 leaflets to the plasma membrane and also need to think of how the 2 leaflets are oriented as the process is occurring o Another example of drawing a siple illustration would help to know the orientation o Exocytosis and endocytosis functions at the plasma membrane to move componets into the cell and move compnoents between the plasma membrane and the vesicles Slide 11 - Vesicles are small membrane enclosed organelles in the cytoplasm of a euk cell - They don’t have a specific functions in terms of what’s going on in the cell but their function is to move components around in the endomembrane system - Ex: Shuttle stuff from endoplasmic reticulum to the golgi - The compartment where the vesicle is coming from is called the donor compartment - Vesicle buding off from the donor compartment and that transfers the content from the donor compartment into the lumen of the vesicles and transfers lipids from the bilayers from the donor compartment into the vesicle as well as any membrane associated proteins from the donor compartment into the vesicle - Then the vesicle can fuse with the target compartment - And that fusion delievers the contents from the lumen of the vesicle to the lumen of the target compartment  also delievers components of the vesicle into the membrane so the phospholipids as well as the proteins - Notice: throughout this process the proteins maintain their orientation with the diff membranes - Part of the protein that protruding into part of the lumen in the donor compartment is also protruding into the lumen of the vesicle and also into the lumen of the target compartment - Part of the protein protruding into the cytosol stays protruding into the cytosol throughout the transfer process - This way in which the protein is associated with the membrane is a result of the way in which the protein was originally inserted into the endoplasmic reticulum when it was being synthesized - And that protein symmetry is maintained throughout this process of circular transport and in that way the protein can arrive at its final destination in its proper functional orientation - Eg: na k pump: it needs to pump na out and k in. And the ATPase domain is in the cytosol so it can access ATP in the cytosol to drive the process. the way in which all these vesciles occurs ensures that the protein ends up I the proper orientation. Slide12 - How are spstific proteins targeted to different orgenelles? o 1 step for synthesizing any protein mRNA arrives in the cytoplasm and translation starts on ribosomes in the cytosol o For a cytosolic protein it’s translated ENTIRELY in the cytosol o It doesn’t have a sorting signal and this can be considered a default location Slide 13 - The way in which proteins were sorted into mito and chloroplasts o Although mito and chloro has their own genome and their own ribosomes, most of their protein is nuclear encoded so the proteins need to enter into these organelles. o Proteins enter the cytosol and they’re targeted by a signal sequence o And they’re imported into the orgenelles o And this sorting is POST TRANSLATIONAL o Proteins remain unfolded in the cytosol o With the association with HSP70 (a chaperon) so that’ll keep the proteins unfolded until they enter the correct location in their destination organelles  Post translational happens after translation Slide 14/15: path of a secreted protein from translation to secretion - But as translation is still occuring insertion of the growing polypeptide chain occurs into the endoplasmic reticulum  called cotranslational process (happen while translation is occuring) - It’s a signal sequence on the protein often at the amino terminal end that targets the growing chain to the ER - As soon as the signal sequence is translated, the signal sequence then gets the growing chain into the ER and transfer across the lipid bilayer of the ER starts in a cotranslational manner - For this process to occur to occur that signal sequence is recognized by a protein called sorting receptor  targets the sequence to the ER - This signal sequence one’s it’s targeted to the ER it interacts with a transport protein of the ER  protein translocator  this protein translocator is used to transfer the protein across the membrane and the signal sequence is what allows it to do that - As the ribosome translates the mRNA the growing polypeptide chain continues to be transferred across the lipid bilayer by this protein translocator - Once enough of these polypeptide chain is synthesized then another protein called a singal peptidase cleaves that signal seq from the protein (in case of a secreted protein), cleaves the protein and now the protein that’s passed through the translocator once it’s completed, it’s no longer associated with the protein translocator or with the plasma membrane and it ends up in the ER lumen - One mRNA can be translated by more than one ribosome at a time…many proteins can be made from one mRNA rapidly. When the ribosomes are finished translating the mRNA.. they dissociate away from the mRNA and return back to the cytosol  ribosomal subunits find another mRNA and start translating that mRNA into protein. if it was a protein that’s going to be targeted into the ER this process would start again - Protein now has been translated and transferred by that protein translocateor across the ER membrane as it was being translated the sequence was cleaved and the mature one is in the ER lumen now it has to be secreted to the extracellular fluid Slide 16: pulse chase experiment - Used pancreatic cell b/c they make lots of secreted proteins - Easier to follow the path of protein because there were so much pancreatic proteins being made in the pancreas - Provide the cell with a short pulse of radiactove amino acids and that pulse of amino acids was followed by a chase of nonradioatice amio acids - The scientists were able to follow the path of the radioactive amino acids as they were encorporated into proteins - Then they were able to follow the path - This is called as a pulse chase experiment: - Pulse of a label is given (radioactive amino acid) and it’s chased away with unlabelled amino acids and you follow that pulse as it moves through the cell - The pulses showed the silvery colored lines, and then they concluded that One of the first places the secreted proteins were targeted was into the rough ER Slide 17: proteins move from the.. - Red dots = location of the labeled protein - At a short chase time(3 mins) the labelled proteins were found in the rough ER - Now we know that occurred from them being targeted and cotranslationally being translated across the membrane of the rough ER into the lumen - With longer chase time (20 mins) they were found in the golgi apparatus and no longer found in the rough ER but they were found as a part of the golgi appartus - After even a longer chase time (120 mins) found in secretory vesicles found in the plasma membrane and could be visualized undergoing the process of exocytosis - This experiment revealed the path of secreted protein within the cell. After ging through the RER it goes through the golgi and then exocytosis. - Doing this scientist demonstrated the temporal link b/w those compartments (components that were first part of the ER was later visualized in other compartments of the cell in an organized manner Slide 18: secretory pathway - That described the secretory pathway  proteins that are produced are continually secreted - Constitutive pathways: o So once the protein is made an it makes it way to the ER and the mature vesicles arrive close to the plasma membrane and then the fuse with the plasma membrane and the contents are secreted  Ex: digestive enzymes  Collagen part of the extracellular matrix  These components are a secreted by the cell to form the extracellular matrix around cels - Regulated pathway: o Where proteins are sorted and then once they get through the golgi complex then their stored in the secretory granules close to the plasma membrane where their ready for the export in response to a specific stimulus  Ex: neurotransmitter  ready to be secreted but it’s not secreted until a specific signal is received by the cell that tells it to secrete the neurotransmitter vesicles. Where the neurotransmitter is been made is ready to be leaked, but doesn leak until the signal is reached. - Clicker question: o You are investigating the ribosomes and you’ve developed a technique to label only the rough ER associated ribosome in the beginning of the experiment. Where do you expect to find those labelled ribosomes 30 mins later?  The cytosol and the golgi  this is not the path of a protein the question is asking for the path of the ribosome  those ribosomes in the cytosol once they associate with an mRNA and a polypeptide tarts to emerge  if that polypeptide has a signal sequence on it then the ribome is targeted to the ER but it stays on the CYTOSOLIC SURFACE ON THE ER and it’s the protein that gets transferred to the ER and it’s the protein which gets transferred across the plasma membrane the ribosome doesn’t actually enter the endomembrane system it just coats the cytosolic side of the ER. So the protein might end up in the golgi, but once the ribosome finishes translating the protein it goes back to the cytosol  The answer is the cytosol and the rough ER  If you label ribosomes that are coating the rough ER those ribosome can continue translating the mRNA until they’re finished and cycle back to the cytosol and pick up another mRNA  there’s a continual movement of ribosome b/w the cytosol and the rough ER dynamically moving b/w the 2 locations Slide 19: protein sorting mechanisms: - 3 main types of protein sorting that occur in the cell: o Process of gated transport ocurs in proteins that needs to get from where they’re synthesized in the cytosol in the nucleus. So proteins move from the cytosol and the nucleus through a nuclear pore complex (NPC)  large complex of proteins that make a pore across the nuclear membrane. (recall: nuclear mem is a double membrane) so makes a pore across this double membrane and that pore moves proteins in and out by a regulated manner (why it’s called gated)  depending on the protein it might be allowed through the gate or not allowed through the gate o Transmembrane transport protein translocators are needed to transport specific proteins across the membrane and that happens for different organelles  Translocaters that insert to the ER co translationally, tere are translocaters which insert into mitochondria, peroxisome etc., these are post translational o Vesicular transport where membrane –enclosed transport vesicles ferry proteins from one compartment to another and the proteins they’re ferrying might be in the lumen of the vesicle or a transmembrane protein that crosses the membrane that’s associated with the lipid bilayer of the vesicle. Those vesicles are needed to get the protein from the ER to other parts of the endomembrane system.  because of all tj proteins made inside our cells this process needs to be very regulated due to the wide range of proteins available in the cell. Slide 20: signal sequences: - One of the ways proteins are regulated - These are stretches of amino acid sequences of a protein that directs a protein to the correct location of the cell and each signal seq speciefies a sepcifc destination. o Signal sequences for nuclear import and export o Signal sequences that direct proteins to the mitochondria to ER, to peroxisomes and other orgnelles - These signal sequences are recognized by other protein, by sorting receptors, and these are that take that protein and helps it get to its correct destination depending on the signal sequence Slide 21: video of protein sorting to the ER - Recapped how protein is first targeted to the ER - And translocated cotranslationally across the ER membrane and this protein would end up in the lumen of the ER when that signal sewuence was cleaved from the amino terminal end Slide 22: signal sequences: - Are oftern found at the N-terminus of the protein (amino terminus) Slide 23: sorting of a secreted protein: - Signal peptidases: can removes the signal sequence of the protein. It can also be internal stretches of AA which remain part of the protein. - The signal sequence is hydrophobic so it intereacts wih the hydrophobic interior of the translocator - That’s a cotranslational process that allows the protein to be passed through the membrane of the ER into the lumen which happens via a protein translocator. Signal sequence is cleaved left behind in the ER membrane secreted protein ends up In the ER lumen  Signel sequences are eventually degraded by the proteins  sected proteins moves in transport vesciles via the secretory pathway  released by the Exocytosis at the PM Slide 24: what about the transmembrane protein that need to be inserted into the membrane? - That process also occurs at the ER - Translation starts at the cytosol - Ribosomes bind to the mRNA and start translating - The transmembrane protein still has an amino terminal (N-term) signal sequence which is also called a start transfer sequence (starts the transfer of the protein across the ER membrane through a protein translocator) - Similar to the last example this amino terminal (N-term) sequence starts the transfer of proteins across the protein translocator and as translation continues more and more of the protein i
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