Cell Bio Exam #2 Study Guide PDF

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Cell Bio Exam #2 Study Guide 1. Membrane Biological Functions a. barrier for diffusion - diffusion: movement of molecules from high concentration to a low concentration - allows some molecules molecules to move freely across while blocking others from moving across - the only was to see what will cross the membrane is to do an experiment to where you add a certain solute to one side of the membrane and see if it ends up on the other side of the membrane - easily move through the membrane  O2  CO2  H2O  Steroids - not easily diffused through the membrane  Ions (because they are charges and are extremely attracted to the water on the outside of the membrane)  Glucose (big)  Glycine (a.a)  AT 

2. Methods of Traveling Through Membranes A. Diffusion - is the movement of a substance from an area of high concentration to low concentration - O2; travels down it’s concentration gradient

B. Facilitated Diffusion - uses carrier proteins to transport specific substances across the membrane

C. Ion Channels - are transport proteins that allow ions to enter the cell - Na+, K+, Ca2+, Cl-

D. Osmosis - is the diffusion of water through a selectively permeable membrane from an area of high concentration to and area of low concentration - aquaporin’s: protein channels through which water can travel across the membrane

E. Active Transport - the movement of substances across the membrane against its concentration gradient (low to high), REQUIERS INVESTED ENERGY ATP - carrier proteins can be used but function as a pump instead because substance is moved from and area of low concentration to an area of high concentration - ex: sodium-potassium pump - there are two types of active transport 1. Primary Active Transport - coupled to ATP to produce ADP +Pi - Ex:

2. Secondary Active Transport - the energy comes from the concentration gradient - only works if the concentration gradient is maintained - Ex:

3. Proteins a. The Pulse Chase Experiment - Main Question: How do proteins get where they need to go? - they took guinne pigs and injected the with radioactive a.a., the reason they were radioactive is because they wanted to be able to track the a.a - the idea was if the put radioactive a.a. acids in the blood some of the a.a. would be made into new proteins, then they added the radioactive a.a. they added a bunch of nonradioactive a.a. in order to see where the radioactive a.a. ended up and to have a cut off - ** pulse = flooding the blood with radioactive a.a. - ** chase = adding the nonradioactive a.a. to the blood -they took cross section of the pancreases because the pancreases is know for it’s secretions - the results 1. within a few minuets of pulsing they saw radioactive proteins associated with the rough endoplasmic reticulum (rER) 2. about 20 minutes later they saw radioactive proteins had left the rER and had travel to the Golgi Apparatus 3. about an hour latter the radioactive proteins are associated with the zymogen granule, which is a secretory vesicle 4. the guinne pigs were fed after the pulse and chase and the radioactive proteins disappeared, no longer visible **** this experiment told scientist that proteins that are secreted out of the cell are made in the rER and then travel to the Golgi Apparatus and then travel to a secretory vesicle b. Zip-Code Hypothesis - a zip-code “signal sequence” tells proteins where to travel when traveling out of the cell - only found all proteins that are traveling 1. out of the cell 2. imbedded into the cell membrane 3. the Golgi Apparatus, rER, Lysosome - located on the N terminus of a protein - the Experiment - they took a bunch of proteins and started chopping off varying sections of the N terminus on one bunch of proteins and varying sections of the C terminus off another bunch of proteins - they found that they proteins with any portion of the N terminus chopped off did NOT travel out of the cell like they were suppose to - this shows that the whole portion of the N terminus is needed for the protein to be able to travel out of the cell c. How do proteins move across a membrane - first off the ribosomes responsible for making proteins are found in the cytoplasm of the cell and on the rER, the proteins located on the rER are only transient there they will fall off once they are done making their protein - for all proteins that are going to exit the cell synthesis starts in the cytoplasm and is then stalled and will the finish on the membrane of the rER - SRP protein: Signal Recognition Protein - binds to the newly synthesized protein, STALL TRANSLATION - on the rER membrane there is a signal receptor for the SRP call SRPR - will move the stalled translation complex to the rER and this means the stalled translation complex is now docked to the membrane of the rER - only binds to proteins that will be secreted from the cell - when SRP binds to the SRPR this causes the SRP to let go of the protein synthesis complex and then it binds to the translocon via the signal complex - translation is started again when SPR lets go

- the enzyme that cuts the signal peptide is called Signal Peptase; this is the reason proteins that are secreted out of the cell are around 20 a.a. shorter then their predicated sequence - B:P - B:P is bound to the inner membrane of the rER - is a chaperon protein because the protein is not folding as it is being synthesized instead its being transported across the membrane through the translocon and then it is folded; and B:P is there to stop the unfolded proteins from interaction with each other through their nonpolar regions - B:P can act as a sensor protein by two ways 1. when B:P is not bound it becomes a transcription regulator; which in turn increases the amount of chaperon proteins being produced 2. when B:P lets go of the sensors they become active; this becomes phosphorylated which effects protein synthesis by blocking translation which means the cell can catch up on the unfolded proteins and fold them better because translation is stalled

D. How does a Protein Become Embedded in the Membrane - first off the ribosomes responsible for making proteins are found in the cytoplasm of the cell and on the rER, the proteins located on the rER are only transient there they will fall off once they are done making their protein - for all proteins that are going to embed in the membrane synthesis starts in the cytoplasm and is then stalled and will the finish on the membrane of the rER - SRP protein: Signal Recognition Protein - binds to the newly synthesized protein, STALL TRANSLATION - on the rER membrane there is a signal receptor for the SRP call SRPR - will move the stalled translation complex to the rER and this means the stalled translation complex is now docked to the membrane of the rER - only binds to proteins that will be secreted from the cell - when SRP binds to the SRPR this causes the SRP to let go of the protein synthesis complex and then it binds to the translocon via the signal complex - when SRP binds to the SRPR this causes the SRP to let go of the protein synthesis complex and then it binds to the translocon via the signal complex - translation is started again when SPR lets go - however once the hydrophobic stop transfer sequence is read the translocon is opened and the protein is moved into the membrane and then the rest of the protein synthesis is completed - the protein is imbedded in the membrane multiple times then there are multiple hydrophobic stop transfer sequences that causes the translocon to open up many times - the enzyme that cuts the signal peptide is called Signal Peptase; this is the reason proteins that are secreted out of the cell are around 20 a.a. shorter then their predicated sequence - normally imbedded proteins have the N terminus on the outside of the cell and the terminus on the outside = TYPE 1 - proteins that have their N terminus on the inside of the cell and C on the outside are know ad TYPE 2

4. Animal Cell Organization a. Motor Proteins 1. Kinesins: move from the inside of the cell outwards 2. Dynesins: move from the outside of the inwards

b. Calnexin - a similar sugar structure added to all proteins made in the lumen of the rER - chaperon protein - decreases the rate at which proteins interact with each other - sugars allows for binding and unbinding; 3 things can happen when it unbinds 1. ultimately packaged into vesicles and travels to the Golgi 2. further modification so it rebinds; this increase the amount of time the protein will spend in the rER; however proteins tend to get kicked out of the rER to the Proteasome after a while 3. will travel through rER membrane into the cytosol and to the proteasome **** a lot of energy is spend in these processes - the cell posses the ability to ramp up correctly folded proteins made in the rER c. Where do proteins go once they are folded - Endomembrane System - almost all proteins that exit the rER travel to the Golgi Apparatus

5. The Golgi Apparatus

- the Golgi apparatus dose far more then just shipping proteins all over the cell - what we know: 1. the sugars that are added in the rER get modified in the Golgi 2. Sugars are also added in the Golgi - Golgi Apparatus Time Line - Rough ER  Golgi Apparatus  Cell Surface or Lumen - Golgi Apparatus Time and Space in the cell  Rough ER Golgi Apparatus  Lumen  Cell Surface a. Golgi Apparatus Arrangement a. Cis Face: where the Golgi and the ER come together b. Trans Face: things exit the Golgi - there are particular sugars added in each stack; meaning each stack had a different biochemistry

b. 2 Proposed Mechanisms of the Golgi Apparatus 1. Vesicular Transport System - vesicles fuse and un-fuse to each stack and each stack adds and modifies sugars - here it is important to make sure the vesicles don’t take the material that separated the cis stack from the medial and trans stack so they can all maintain their unique biochemistry

2. Cisternae Maturation - cis-stack will form to a medial stack and a medial stack will form a trans stack and the trans stack breaks down into vesicles - here vesicles moving in the retrograde direction carry the unique “stuff” of each stack to take it back to the new stack forming in order to maintain the stacks unique biochemistry - the vesicles coming fuse together and become the new Cis-stack

Ex: Collagen Fibers

- if you look at cells creating collagen fibers the fibers are larger then the vesicles being formed this means that in vesicular transport the fiber would have to be broken down before its moved to a mew stack, where as in cisternae maturation the collagen fibers wouldn’t have to be broken down because the stack will move forward and become a new stack - Why are both mechanisms considers as plausible? - because there have been vesicles found moving the forward and backward direction - THE MOST ACURATE MODLE INVOLVES BOTH OF MECHANISMS - Big proteins move slowly  Cisternae Maturation - Small proteins move slowly  Vesicular Transport 6. Vesicular Movement From the rER to the Golgi Apparatus a. GTPase-Protien - a protein that breaks down GTP into  GDP - when the G protein is bound to the GTP this protein can bind to other proteins and control what they can do, this is considered its “on” position - when a G-protein is bound to GDP this protein can not bind to other proteins this is considered it’s “off” position; it will kick out the GDP and rebind it to a new GTP - GAP: increases the rate at which the G-protein goes from on to off -GEF: increases the rate at which GDP is kicked off and increases the rate at which the Gprotein goes from off to on - Are ALWAYS considered on when they are bound to the membrane

b. Vesicular movement from the rER to the Golgi - SAR-1 (G-protein) will be in the “off” state and then once it binds to the GEF on the rER membrane it will be switched to the “on” state; the GEF ends up in the vesicle - once SAR-1 one binds to the membrane COP II proteins will bind to the membrane around SAR-1 and over SAR-1 and will pull the membrane out into a bulge - once the vesicles has been formed and is en-rout to the Golgi (via dineines) the COP II act like a GAP making SAR-1 turn off and then the COP II proteins and SAR-1 protein will fall off from the vesicle letting it interact with the membrane - there can be two ways that proteins make it into the vesicle 1. the proteins that need to exit the cell will bind to the cargo receptor found in the rER membrane between the COP II proteins 2. the proteins get caught up in bulk-flow

7. Vesicular Movement from the Golgi to the rER

- ARF-1 (G-protein) is considered “off” when it is not bound to the rER, but it bind to the GEF on the membrane it is them considered “on” - the GEF that is used is a different GEF then the one on the rER membrane - once ARF-1 is bound to the membrane then COP-1 proteins come and bind to it and the membrane around it and make the membrane bulge outwards - once the vesicle is en-rout to the rER (by kinesis) the COP 1 proteins act like a GAP making the COP I proteins fall off allowing the vesicle to interact with the membrane

a. How Do Proteins Make It Into The Vesicle? - there are cargo receptors in the Golgi membrane that have KKXX signal sequence at their C-terminus; which are swept into the membrane as the vesicle forms - these cargo receptors have to have the signal sequence (KKXX) so they are placed in the membrane - the KKXX sequence interacts with the COP I proteins - the part of the cargo receptor that is on the inside of the vesicle interact with proteins that contain the KDEL sequence - the KDEL sequence indicates that they protein needs to go from the Golgi to the rER - can be found anywhere in the protein - residential rER proteins contain the KDEL sequence - the protein probably got swept up by bulk flow into a vesicle

8. Movement of a Vesicle from the trans-Golgi to the Lysosome

- Things that typically go to the lysosome go there to get degraded by enzymes - the lysosome had a pH of 2, which is really acidic, so the proteins are denatured and so that the protein that work in the lysosome will only be able to work in the lysosome this way if they make their way out of the cell they will be unable to degrade other proteins - when ARF-1 is not bound to the membrane it is considered in its “off” state and when ARF-1 binds to the membrane of the trans Golgi it is considered to be turned “on” - once ARF-1 binds to the trans membrane of the golgi then Clathrin (coat protein) will come and bind to the membrane and bind around ARF-1 - the proteins that need to travel from the golgi to the lysosome will contain a manos-6 sugar structure; this sugar structure is added in the in the golgi apparatus - there will be a manos-6 phosphate receptor that binds to the proteins contain the manos-6 sugar structure

9. Differences in Vesicular Transport

Type of Coat Protein Type of G-Protein Direction of Movement Cargo Receptors

Golgi  rER COP II SAR-1 Retrograde Contains the KKXX signal sequence at it’s C-terminus which will interact with the KDEL sequence

rER  Golgi COP I ARF-1 Anterograde Contains cargo receptors

a. Proteins that are NOT made by a ribosome bound to the rER 1. ARF-1 2. SAR-1 3. COP I 4 COP II ** These proteins are made by a ribosome in the cytosol b. Where will these proteins end up? 1. A protein that contains no signal sequence  cytosol 2. A protein that contains a signal sequence  secreted from the cell 3. A protein that contains a signal sequence and a KDEL sequence  rER 4. A protein that contains just a KDEL sequence  cytosol

10. Vesicle Fusion - vesicles will fuse with target membranes

Trans Golgi Lysosome Clathrin ARF-1 Anterograde Manos-6 phosphate receptor that binds to the manos-6-sugar structure

- there are 2 classes of proteins that will help with this fusion 1. RAB proteins - GPase protein - found on the membrane of the vesicle and found on the target membrane; need to have compatible RABS - “Lock and Key” method - is considered a G-PROTEIN

2. SNARE Proteins - there are snare proteins on the membranes of the vesicles that are being transported called VSNARE - there are snare proteins on the target membrane are these are called T-SNARE - as snares interact with each other they undergo a conformational change and break 2 wholes in the membranes so the vesicle can fuse with the target membrane - ARE NOT G-PROTEIN

11. Cell Signaling - cells know what goes on outside of them and they change what they are doing in response - can also be called signal transduction a. Switches 1. The GPAse Switch -when the G protein is bound to the GTP this protein can bind to other proteins and control what they can do, this is considered its “on” position - when a G-protein is bound to GDP this protein can not bind to other proteins this is considered it’s “off” position; it will kick out the GDP and rebind it to a new GTP - GAP: increases the rate at which the G-protein goes from on to off -GEF: increases the rate at which GDP is kicked off and increases the rate at which the G-protein goes from off to on

2. Kinase - ATPase’s that break down ATP into ADP and an inorganic phosphate group

- changing the protein structure and there for can be changing it’s function by turning it “on” or “off” - important because you’ll amplify the signal - cascading signals can interact with other signals at nodes, because the signals are interconnected this allows for signal integration

b. What are signaling molecules? 1. Proteins 2. Fatty Acids 3. Ions 4. Amino Acids *** signals can be diverse chemically and structurally!! c. Membrane Soluble Signals - this means the signal can pass directly through the membrane and will directly bind to the receptor - the receptor is called: Nuclear or Intracellular Receptor - these receptors are highly selective - the signals that can move through a membrane are a. estrogen b. Testosterone c. Progesterone

d. Signals That are Not Membrane Soluble - they work through Signal Transducers and there are two types of Signal Transducers 1. GPCR: G-Protein Coupled Receptor

- in the cell membrane there is a G-protein Receptor (7TM; crosses the membrane 7 times) and there is a htGPAse complex (contains and alpha, beta and gamma parts) - these sense molecules outside the cell and activate signal transduction inside the cell

- alpha beta and gamma are all bound together when alpha is turned “off”, alpha separates when alpha turns on - when the signal comes and binds to the G-Protein Receptor then the htGPAse complex moves and binds to the C-terminus - once it binds to the C terminus then alpha turn on a separates from the rest of the complex (beta and gamma) and goes and binds to the effector - once alpha binds to the receptor this turns the transduction cascade on and then turns something “on” in the cell - once alpha binds to the receptor GAP turns alpha off causing the signal to stop being sent

i. The Effector - there are multiple effectors - what the effectors is depends on what alpha is - there are three main types Gαs

Gαi

Effector

Adenylyl Cyclase

What dose it do?

Take ATP and converts it to cAMP Turns on Adenylyl Cyclase

Adenylyl Cyclase Takes ATP and converts it to cAMP Turns off Adenylyl Cyclase

Effect

GαQ...