• Study of structures within a cell and what/how they work
• How these cells create the structure of the body

• Protein localization
• Membrane trafficking
• A cytoskeleton

• Cell cycle
• Cell adhesion

Cancer can destroy these functions

• Proteins must be targeted to specific locations within a cell
• The sorting signals are amino acid sequences with proteins that direct protein localization and transport (basically they are a "zipcode" that tell a protein where to go)
• They are found at the N-terminus

• They can range from 2-60 amino acids long
• They are found at the N-terminus

• Peptidases cut the amino acids off after the proteins reach their location

1. Molecular Biology Approach
2. Biochemical Approach
3. Genetic Approach

• First, take bits of a transported protein and clone its amino acid to a reporter gene (usually GFP). GFP glows!!!!
• Where the GFP goes, they can follow the glow.
• If the protein is fused to the GFP correctly, it will go where the amino acids tell it, and you can follow.

• Purifying the signal sequence
• If it goes to the right location it gets clipped off
• Somehow this tells us something

• Break specific parts of the protein to identify the sequence for localization
• Keep breaking off parts until the gene stops localizing. When it stops, you know the last part you broke off was the essential part

1. Passively diffusing to correct locations
2. Transport

Of protein transport, this can include...

1. Gated Transport
2. Translocator-based Transport
3. Vesicular Transport

• This is the controlled opening of pore complexes
• Mainly occurs into and out of the nucleus

• When transmembrane protein translocators transfer proteins from one side of the membrane to another; the proteins must unfold to pass through
• It occurs in the mitochondria, ER, peroxisomes and plastids from the cytosol

1. A few proteins are made in the mitochondria and directed by mitochondrial DNA, but most are imported from the cytosol post-translationally.
2. Most proteins destined for the matrix have a cleavable N-terminal signal sequence
3. The matrix symbol is usually an amphipathetic (one hyrophobic one philic end). The hydrophobic end bonds to the matrix importer!

Transport into the matrix. It requires transporter complexes (TOM and TIM), protein chaperones (Hsp70) and energy (ATP and membrane potential)...

1. When the protein binds to the TOM complex, Hsp70 (chaperone) keeps it unfolded. The protein then starts moving through the membrane. Displacement of translocator and chaperone begins with an ATP dependent process.
2. On the inner membrane, the inside is positive and the outside is negative. Since the signal sequence is positively charged, it wants to get through.
3. The signal sequence is removed by a mitochondrial protease. As the protein emerges, Hsp70 binds to it and ensures directionality. After it is finished emerging, Hsp70 removes itself and the protein folds.

• Keith Porter in 1940
• It makes up 50% of membrane surface (lots of surface area)
• It continually forming, breaking down and reformeing again.
• It's continuous (one membrane all the way around and stuff)

1. It is the entry point/start of the membrane trafficking membrane (to anywhere)
2. Performs quality control of proteins
3. It performs protein modifications
4. Stores intracellular Ca+
5. Location of lipid biosynthesis (psht whatever that is)
6. Detoxification (of like alcohol and things ha)

1. Smooth like a classy guy
2. Rough like a gangster

• Made up of shorter tubules
• It stores calcium (especially in the muscle cells)
• It leads the production of lipids
• Where the detox enzymes are

It can vary in size for different cell types and expand when needed (transformer!)

• Longer, flat stacks
• It's rough because of all the ribosomes that attach to the membrane
• It is the cytoplasmic entry point for proteins into the vesicular trafficking system
• Transport of proteins into the ER is co-translational (like co-transport, uses the energy of protein translation to translocate it into the ER)
• Modifies dem proteins yo

• Well collagen is the main protein in connective tissues, but they need modifided amino acids to function? Maybe I don't know.
• These modifications of the amino acids are performed in the ER.
• Somehow not having vitamin C fucks this up, maybe the ER needs it or something. I don't know.

I was not paying attention during this slide obviously so I really have no clue how any of this is connected.

• Translation is the process of an mRNA attaching to a ribosome. The ribosome reads the mRNA as it passes along, creating a protein from the start codeon to the stop codeon.
• Two pools, one free floating and one agains the ER membrane.

• RNA has ATG (start signal), the ribosome (enzyme) and the stop signal (TAA, TGA or TAG)
• The ribosome adds amino acids together, making a protein, until it reaches the RNA stop signal
• The energy of this translation is used to push the emerging protein in the ER at the same time (co-translational transportation)

• It's basically one big protein that's made up of 6 different proteins put together. It has an RNA subunit that is long and linear so it can "reach" out and "grab" stuff
• It possesses a critical GTPase protein (remember GTPase from cell signaling?) When GTP is bound to it, it's active. When it hyrdolyzes it into GDP, it turns to inactive
• General Ftn: It binds to signal sequences... why?

• The RNA subunit gives it the ability to wrap and bind to rRNA and proteins, causing a pause in translation.
• The ER has SRP receptors that it will bind to while in the position.
• When it has bound to the SRP receptor/ER, it triggers the GTPase and the release of the SRP. Translation resumes
• The signal is then cleaved off

Basically a way to make sure that a protein in translation can find its way to the ER before it's completely translated.

• The start signal is recognized by the SRP and translocator.
• The translocator can open two directions- towards the cytoplasm and sideways, into the membrane. This signal sequence (6-12 amino acids) tells it which to do! More no next card...

• Each one can tell the translocator to do one of two things, push them through the membrane, or keep them within. After this, they can say one of two more things, cleave me or let me stay!
• Of these two options, the protein can use different combinations. The start signal can say hey, keep me in the membrane, and then cleave me when I'm done. When the stop signal comes through, it can say keep me in the membrane too!
• Or both can come through, say to stay in the membrane, and asked to remain attached. 

• They cannot be cleaved! This helps create double pass proteins. Look at the slide, visualization helps.
• Double pass is basically a protein with two or more ends on both both sides of the membrane

1. Lipidation: adding a fatty acid on
2. Hydroxylation: adding an -OH group
3. Disulfide Bond Formation: adding a cystein amino acid (-CH2-S-S-) in place of the R group
4. Glycosylation: adding a sugar/carbohydrate

It is a protein that is anchored to the ER inner membrane (lumenal) by lipids

• Certain proteins that are translocated through have a C terminal peptide (start/stop signal ends in COOH on opposite end).
• When it gets cleaved off, the free end of the protein is hydrophobic, so it finds hydrophobic friends so it's not lonely
• These anchors are what it bonds too, and they are GPI linked to the membrane.

Remember those lipid rafts? These are what they are/how they're formed!!!! Groups of GPI anchored proteins!!!

• Lipid rafts: localization of proteins together can help favor certain reactions and signals
• Acetylcholinesterase (very fast enzyme) is anchored, this helps it be so fast and what allows quick movement
• Can be cut by phospholipase (trypanosomes do this to avoid detection by the immune system; African Sleeping Sickness)

• In the ER AND the Golgi
• A 14-sugar oligosaccharide is attached
• It's N-linked because it is attached to a nitrogen off an Asparagine side chain on the protein
• Oligosaccharide transferate mediates the transfer! How? The transferate is integral (transmembrane) with an active site on the inside. As a protein is being translocated through and the Asparagine chain passes by it, it will break a bond and attach this oligosaccharide.
• The energy comes from the breaking of a pyrophosphate bond when attaching the oligosaccharide.

In the long run, proteins HAVE to fold.

• Hydrophobic amino acids want to collapse and be near other hydrophobic amino acids. However, up the 80% of proteins don't fold right and need to try again or be degraded.
• This oligosaccharide and the glucose it has helps a cell mediate this protein folding. Will see this in next cards...... 

LONG CARD BE READY YO

How does the addition of an oligosaccharide help the ER control misfolded proteins?

1. Upon entry into the ER, 3 glucose are immediately trimmed off. When proteins are folded correctly, they stay this way. If a protein is folded incorrectly, glucosyl transferase finds them and addes the 3 glucose back on. They are able to find them because misfolded proteins have an exposed hydrophobic end.
2. CALNEXIN: this binds to the proteins in the ER that have the 3 terminal glucose and retains them in the ER, giving them more time to fold.
3. GLUCOSIDASE: this floats around and removes glucose. If the glucose are removed but the protein is still not folded correctly, the process can restart. If the protein is finally folded correctly, no glucosyl transferase can attach glucose, which means Calnexin cannot retain it, and the protein will leave. YAY!!!!!

• Each oligosaccharide has a mannose on it.
• In the ER, there are mannosidases floating around. These enzymes are EXTREMELY SLOW ACTING (slower then glucosyl transferase, calnexin and glucosidase).
• On the previous card, a protein is given many chances to unfold and try again. But sooner or later, mannosidase will cut off the mannose.
• When this mannose is cut off, it is a sign that the protein just needs to be sent to the cytoplasm to be degraded.
• In the cytoplasm, when a protein has an N-linked glycosylated group, this is what tells it to degrade it.

1. Cell creates more ER to deal with the problem
2. Cell decreases rate of translation (via slowing down ribosomes) until it is caught up
3. Cell increases the number chaperone proteins. These proteins completely unfold proteins and then allow them to attemp the process again.

• Exocytosis: trafficking proteins to the cell surface
• Endocytosis: trafficking proteins from the cell surface to somewhere on the inside of the cell

1. Cargo recruitment
2. Vesicle Formation
3. Transport and Targeting
4. Tethering
5. Fusion

• There are cargo receptors on the membrane of the ER that select cetain proteins to be transported! These proteins bind to the receptor and they hold them there. Some proteins don't need to be moved and are held back (ex: calnexin holding proteins)
• Transmembrane proteins that need to be moved are recruited during points of vesicle formation

• As coat proteins gather, adaptor proteins start binding to cargo receptors on the outside of the membrane.
• These adaptor proteins basically help draw and bind even more coat proteins to the forming vesicle, allowing it to former faster.

• The coat proteins cannot cut themselves, they need help.
• Dynamin, a protein, come in and cut the neck of the vesicle that's still attached, allowing it to detach.

Inbetweener step! What problem do these "coats" have? HOW DO WE FIX IT IN THIS TOTAL CLUTCH MOVE STEP?!

What is Sar1 amphipathic?

All of this step happens during vesicle formation (it's a precursor to fixing this problem).

• Coats needs to be removed so the vesicle can be targeted, drawn in and fused by it's target membrane!
• Luckily we have GTPase proteins for this, which is just wondeful. Arf helps take apart Clathrin and COPI. Sar1 helps take apart COPII. 
• The cargo receptor proteins function as GEF in this case because the GEFs active GTPase proteins (which is what Arf and Sar1 are, and they need to be active to work of course).
• Sar1 is amphipathic because it has a nonpolar end that buries into the interior of the vesicle and a polar end that extends out and attaches to other hydrophilic things. This is how it anchors to the vesicle and recruits adaptor and coat proteins. (AKA these attach before scission and help form the vesicle).

• Well, they're GTPase proteins, MEANING that sooner or later they will hydrolyze GTP to GDP!
• When this hydrolization happens, they become inactive, pull in the amphipathic helix and in turn ALL the adaptor and coat proteins fall off. YAY

Pre-cursor to Targeting! (Really there should be more than five steps to this process cause there's like these 6000 inbetween steps and this is all just a hoax so screw it).

What are Rab proteins (what this flashcard is really about)? What the two things they can bind to?

• They are another class of GTPase proteins
• There are over 60 of them and they are basically the address layer for entire vesicles; they tell it where to go.
• They can bind to motor proteins (not important for this test) and TETHERING PROTEINS (YES THIS IS IMPORTANT FOR THIS TEST).

• On the target compartment, there are bound tethering proteins that hang off. They can assemble into long, multi-protein complexes that reach out into the cytoplasm
• These tethering proteins grab onto the rab proteins that are on the vesicle, anchoring the vesicle to the membrane.

• Fusion is lead through SNARES. There are v-SNARES (on the vesicle) and t-SNARES (on the target compartment). The t and v are fairly self explanatory and you should just quit if you can't understand that.
• 1 v-SNARE and 3 t-SNAREs zip together to make a 4 helix bundle.
• They keep zipping and zipping and through the magical POWER of torque, they force H2O from between the two membranes and push them together so hard they mesh and the cargo is released.

• COPII and Clathrin go to the Golgi
• However, cargo receptor proteins need to get back to ER. COPI coat proteins direct them back.

• Same exact concepts from the vesicular transport lecture (WELL LOOK AT THAT)
• There are cargo receptor proteins (lumenal and transmembrane), coat proteins, Sar1 GTPases and adpator proteins
• And not making all those flashcards again so use your brain.

AKA Retrograde Trafficking

1. ER proteins that were sent to the Golgi have signals on them that say "send me back to the ER yo"
2. The ER has resident proteins that act as retrieval signals for these other proteins
3. PROTEINS EVERYWHERE! There's 8 kajillion proteins everywhere and they will be explained in the next cards

1. KKXX: these are located at the C-terminal and have a simple function: the COPI coat proteins bind directly to them; they retrieve resident membrane proteins
2. KDEL (sounds like a type of steak): also at the c-terminus, they are in the lumena, therefore they also have KDEL receptors; they retrieve resident soluble proteins.... yeah

I don't understand this very well, but apparently even though we have KDEL receptors in the ER, we don't want to use them? What? Why? He didn't explain this. Just know this following concept...

To fix this problem that makes absolutely no sense to me...

• KDELs can only function at certain pH's. The pH in the ER is neutral, making all the KDEL receptors inactive, unable to bind KDELs.
• In the Golgi, the pH is acidic, making KDEL receptors active and able to bind KDELs.

So there.

• The KKXX/KDEL system (even though apparently KDELs don't work in the ER WTF I DON'T GET IT)
• Kin Recognition: like proteins recognize each other and assemble into aggregates too large to fit into a vesicle

• It is stacked! These allows more protein modification in which different layers can perform different functions (phosphorylation, glycosylation, sulfylation, etc.)
• It allows even more sorting of proteins on their way out of...
• Cis face: Where proteins enter the Golgi
• Trans face: where proteins leave the Golgi

• The Golgi attaches oligosaccharides and glucose to side chains ending in -OH. Therefore it's O-linked, not N-linked.
• All the Golgi enzymes are membrane bound, allowing easier retention

Overall, just even ANOTHER form of protein quality control. It gives it even more protection/functions.

• Sugars are the most rigid macromolecules. Adding sugars protects proteins from other proteins attaching to them, along with keeping away other organisms
• It keeps the protein from being attacked by proteases. It can also keep bacteria from approaching the cell surface!

1. The Ferry/Vesicular Transport Model: the stacks stay in the place and proteins/vesicles move through these different stacks and compartments
2. The Lava Lamp/Cisternal Maturation Model: Entire stacks (cisternae) transity through the Golgi, carrying the same proteins the whole time. Vesicular tubular clusters support this- as they move towards the golgi they form these compartments that move through as a whole.

They are the nomnomnomers of the cell (garbage disposals).

• Proteasomes can only degrade proteins, which then diffuse into the cytoplasm to be reused
• Lysosomes can do that PLUS degrade many types of macromolecules

• Well, they're just a great mix of everything. First, "material" (I'm assuming we don't need to know specifically what cause he never said what) is endocytosed from the cell surface.
• This stuff becomes an early endosome.
• Then the Golgi is like 'HEYOOO" here's some lipids, proteins and shit. Then the ER is like hey here's some other autophagosome shit!
• All of this stuff from the Golgi just morphs into the early endosome, turning it a late endosome. Then the ER's stuff goops in and turns it into a lysosome.

• Well it varies, but they are some of the most irregularly shaped compartments within a cell
• They have to break down so many different things that these varying, strange shapes help them do this. 
• They breakdown intracellular structures, extracellular structures, phagocytosed cells, bacteria, viruses and nutrients
• They can breakdown macromolecules ot produce new nutrients for the cell

Vacuoles!!!

They store nutrients and waste (in the same place?! unsanitary motherfuckers)

Vacuoles in seeds store proteins

Regulate osmolarity and pH (for when a dog pees on you as Blankenship says)

90% OF THE CELL WTF

• All kinds of destruction enzymes, more spcifically, quite a bit of acid hydrolases. (e.g. lipases, phosphotases, sulfatases, proteases, nucleases, glycosidases).
• These give the lysosome the ability to breakdown almost any macromolecule
• They need an acidic environment to work, this protects the cell because hydorlases don't become active until they're IN THE LYSOSOME. This stops them from breaking things down in every part of the cell. If that were the case, they would just break down the entire cell.
• They use an ATP driven vacuolar H+ pump to pump protons into the lysosome, keeping it acidic.

• Endocytosis
• Autophagy
• Pagocytosis

"Self Eating"

• When a cell component gets worn out or begins to starve (nonspecific), they will go through autophagy
• They get surround by a membrane (creating double cell membrane) which then fuses with a lysosome. Digestion begins.

Follows the same concepts as usual...

• A sugar on the proteins serves as a marker for what it needs. This is mannos-6-phosphate, or "the lysosomal cue."
• This mannose-6-phosphate gets put on IN THE GOLGI. There is mannose on the N-oligosaccharide of the protein (on all of them). On this mannose, there is GlcNac. This GlcNac gets cut off and replaced with mannose-6-phosphate.
• When the M6P finds an M6P receptor, it binds and gets sent to the lysosome!

• M6P receptors function only at a lower pH's and not at higher pH's, which is why they don't just get sent back when they get to the highly acidic lysosome.

1. Nutrient Uptake
2. Maintenance of Cell Membranes
3. Regulation of Cell Signaling

1. Phagocytosis: eating of large vesicle; regulated
2. Pinocytosis: cell drinking; smaller vesicles; contitutive; there are 4 types

1. Clathrin Dependent
2. Caveolin Dependent
3. F-Actin Dependent
4. Dynamin Dependent

The Cell surface is covered in Clathrin coated pits (about 2%). With these, they cell can make clathrin vesicles in less than a minute! There are thousands leaving the surface every minute.

With this process, the vesicles are internalizing membrane and bits of extracellular fluid (hence, cell drinking).

• They are elongated pits in the plasma membrane that are made up of distinct lipids and proteins (caveolins, cholesterol, GPI anchored proteins/rafts)
• They are cut with dynamin and then travel anywhere but the endosome (they like to avoid pH and hydrolase activity); so they go to caveosome

My morale to making these flashcards is quickly fading, I was supposed to be in bed at 2... I can do this.... so.... close....

F-acitin is polymerized which causes it to elongate, creating a bud that eventually just breaks off from the membrane

• This functions to help Clathrin and Caveolin dependent.
• Can also function independently by severing tubules that go into the cell.

Remember early endosomes? Well because they are not fully formed lysosomes yet, the pH is not acidic enough for their hydrolases to function. If there are proteins that can still be used in these, they are transported out and to somewhere else.

With this recyling, proteins can be shuffled around from membrane to membrane, cell to cell, until they can be used again!

Like fusion in vesicular transport, SNARES torque together and fuse a vesicle to the cell membrane. When they fuse completely, the vesicle opens up, releasing its content.

DIABETES YAHHHHH! somehow this has to do with diabetes but he told us it wasn't on the test so I'm not wasitng my precious brainpower with it (there's very very very little left).

• Regulated requires a specific signal/event that causes it to happen
• It transports small molecules (like neurotransmitters) or proteins (sorted for exocytosis from the golgi)
• The molecules start aggregating until they're big enough for clathrin to start budding it. Finally, vesicles come off and package so tight that the contents become crystalline.
• At the membrane, it fuses (with minimal help from SNAREs) and contents are released.
• Can also help increase membrane surface area.