Midterm No. 2, Opus 2 Flashcards
Secondary active transport
Symporters and antiporters
How do cells establish ion gradients?
Primary active transport
How do animal cells establish ion gradients?
ATP-powered pumps ONLY
How do plant cells and some prokaryotes establish ion gradients?
ATP-powered pumps and light driven pumps
How do chemoautotrophic prokaryotes establish ion gradients?
ATP-powered pumps, light driven pumps, and inorganic solute pumps for things like H2S and Fe2+
P-Type pumps
Use ATP to create ion gradients
Unlike other pump classes, this one receives a covalently bound phosphate on an aspartate during its pump cycle. Key feature
It is the only pump with temporary self-phosphorylation. Other pumps can use ATP, but no others receive a temporary covalently-linked phosphate group
Digoxin (the heart medication)
Target is a specific subtype of P-Type sodium-potassium ATPase pump
Digoxin spresses sodium ATPases, which has an indirect effect of making cells worse a pumping out calcium
This is bad if you’re healthy, but great for weak hearts that need the Ca for muscle contraction
Murder mystery connection, spouse may be poisoned with digoxin medication
ABC Transporters
Primary active transport
Largest family of membrane transport proteins
They are ATPases that move solutes UP a gradient
They uses ATP hydrolysis, but there’s no covalent phosphate linkages or modifications like there are in P-type pumps
The energy from the ATP-hydrolysis drives the conformational changes that pushes the ligand out
Family members are diverse. As a family they can transport diverse cargo
How much ATP is used in eukaryotic ABC Transporters?
2 ATP→ ADP+Pi for every exported ligand
ABC Transporter general structure
2 membrane domains, each with 6 TMDs. 2 cytosolic ATP binding domains
Bacterial ABC transporter: importer or exporter?
Importer
Eukaryotic ABC transporter: importer or exporter?
Exporter
ABC transporters in MDR1 cancers
ABC transporters were initially discovered in MDR1 (multi drug resistant) cancers.
Tumor cells that express ABC transporters can pump out more of the anti-cancer and chemo drugs, resulting in patients with MDR1 cancers. It’s a fitness advantage for the tumor cells.
The first ABC transporter ever discovered was the ARCB1 (MDR1), which was overexpressed in a patient’s resistant cancer cells. It was pumping out membrane-permeable chemo drugs.
Mechanistically, ligands can enter from either the cytosol (hydrophilic) or from the cytosolic leaflet (hydrophobic)
Other importances of ABC transporters
Lots of insecticide resistance is related to ABC transporters
The malaria parasite Plasmodium falciparum has developed resistance to anti-malaria drugs by evolving strains overexpressing the ABC transporter PfMDR1
What’s the difference between a symporter and an antiporter?
Relative direction
Symporters
Secondary active transport
Two ions move into the cell
One ion moves DOWN a gradient
One ion moves UP a gradient
The down-gradient ion powers the up-gradient ion
Antiporters
Secondary active transport
One ion moves in the cell, one ion moves out of the cell
Outbound ion goes UP a gradient
Inbound ion goes DOWN a gradient
The inbound ion powers the outbound ion
What kinds of functions/processes need ion channels (i.e. need speed?)
Neural transmission, muscle contraction, cell signaling, and secretion
What’s fastest: ATP powered pumps, transporters, or channels?
Channels, then transporters, then ATP powered pumps
How are channels different from uniporters?
Both are masters of passive transport
Uniporters are like a turnstyle. They need time for their cycle of conformational changes to process before letting another ligand pass through
Uniporters are saturable
Channels are like gates through which a whole crowd can go through, albeit single file. Hence the channel’s great speed
Channels, for the most part, are NOT saturable
Both move things DOWN a gradient
Are ion channels passive or active transport?
Passive
List 4 ways/mechanisms an ion channel can be gated
Voltage gated
Extracellular ligand gated
Intracellular ligand gated
Mechanically gated (the mechanism being things like senses: touch, hearing, osmotic changes causing swelling, etc)
How long are ion channels open?
Only a few milliseconds, very briefly.
A full second of openness would destroy the gradient and possibly the cell
Which occurs faster, an eye blink, an ion channel opening+closing, or a hummingbird’s wing flap?
Ion channel is fastest
Then hummingbird wing flap, then eye blink
ATP synthase
An H+ pump in reverse and a nanoscale rotary engine (rare! not many rotors in nature!)
Subcategories include V-class and F-class synthases. The two classes are structurally very similar, but they rotate in opposite directions and have different jobs
Where are V-class ATP synthases found?
Vacuolar membranes in plants, yeast, and misc. fungi
Endosomal and lysosomal membranes in animal cells
Plasma membranes of osteoclasts and some kidney tubule cells
Function of V-class ATP synthases
Burns ATP to create an H+ gradient
ATP→ADP+Pi occurs in the cytoplasmic side
Where are F-class ATP synthases found?
Bacterial plasma membranes
Inner mitochondrial membranes
Chloroplast thylakoid membranes
Function of F-class ATP synthases
Spends H+ gradients to make ATP
ADP+Pi→ATP happens on the cytoplasmic side or interior membrane space
What is the point of the ETC?
To make a proton gradient across the mitochondria’s inner membrane (remember that the mitochondria’s outer membrane is permeable and has beta-barrel porins in it).
F0 subunit of ATP synthase
Proton-driven motor, rotates. Includes y, e, and c
F1 subunit of ATP synthase
ATP-driven motor, static. Includes all subunits that aren’t y, e, or c
How does the charged H+ ion rotate through the fatty acid moiety in ATP synthases?
H+ binds to c on an aspartate. This neutralizes aspartate’s negative charge, making it easier to spin through the fatty acid moiety.
Describe the flow + transformation of energy in ATP synthases
The H+ gradient is transformed into rotational energy, then mechanical energy, and then to chemical energy when ADP+Pi → ATP
What specifically drives the ATP synthesis in ATP synthases?
y’s shaft differentially “presses” on each and subunit as it rotates. Each and marks a different stage in the ADP+Pi → ATP cycle. The conformational changes within the compressed subunits drive the ATP synthesis
What makes y spin in ATP synthases?
A steep H+ gradient (made with the ETC) is required to drive the process
F0 acts as a merry-go-round, with the aspartate acting as the H+ carrier
Arginine (positively charged) swings between the H+ entry and exit sites. It’s displaced by the incoming proton, which encourages/facilitates the exit of the outgoing proton
When thinking about protein targeting, look for 3 things:
- What is the targeting information?
- Who’s recognizing the targeting information?
- What’s happening to get the protein into the organelle?
Where is ER localized in the cell?
It’s not. It’s actually spread out through most of the cell
That means that if something needs to go from the ER to the golgi, it may not be directly adjacent and may need help to get there
Function of smooth ER
Lipid and steroid synthesis
Assembles most of the cell’s lipid bilayers
Functions of rough ER
Protein synthesis, vesicular transport of soluble proteins (lumen and secreted) and membrane proteins, and calcium storage
Both generic ER and sarcoplasmic reticulums store calcium en masse. Based on this, what kind of transporters or channels should you expect to see in their membranes?
Hella P-type calcium ATPases
Your cell’s cytoplasmic calcium concentration has changed. Why might this have happened?
Either plasma membrane channels have let calcium ions in from the outside
OR
ER channels released some of their calcium store to the cytoplasm
What kind of ER do liver cells tend to have more of?
Smooth ER, for steroid hormone synthesis
What kind of ER do pancreatic cells tend to have more of?
Rough ER, for synthesizing secreted proteins
What kind of ER do muscle cells tend to have more of?
Sarcoplasmic reticulum, for calcium storage used in muscle contractions
How do we know what ER looks like (what experimental techniques were used)?
Transmission electron micrographs
How are proteins targeted to the ER? What experiment was used to reveal this information?
Technique: separation of cellular components via density gradient centrifugation
When ER is mashed up, it breaks into vesicles that reseal (a property of the lipids making up its membrane). This property can be harnessed.
Cells are broken open and the ER is homogenized and then resealed into vesicles
When centrifuged in a sample tube with a sucrose gradient, the vesicles will separate in the gradient based on their density
Smooth microsomes (vesicles containing smooth ER) have lower density and flow up top, in the low sucrose concentration
Rough microsomes (vesicles containing rough ER) have higher density and float lower, in the higher sucrose concentration
Describe early ER visualization experiments
Pancreatic cells were given a bit of radioactive leucine a pulse chase. By conducting the chase at different times, they found they could follow the leucine through the cell’s compartments, tracing it through a protein synthesis journey.
Another ER visualization experiment involves using a viral protein instead of a radioactive one. At high temp the protein would misfold and block the ER. At low temp it would not. When tagged with GTP, it lets us see it travel through the ER, golgi, and plasma membrane.
Do secretory proteins enter the ER lumen? What experimental techniques were used to discern this?
Yes
Experimental technique: give pancreatic cells radioactive leucine (this will label newly synthesized proteins). Then isolate the rough (ribosome-studded) microsomes from the above density separation technique. Treat samples with and without a detergent, then add proteases to all samples
If the proteins are in the ER, then the protease can’t digest the proteins in the samples without the detergent, but will digest them in the sample with detergent (Observed result!)
If the proteins are NOT in the ER, then the protease will digest the proteins in both samples, regardless of detergent presence
Describe an experiment to determine whether proteins are co-translationally inserted into the ER
Run samples of protein synthesis: one sample is in vitro with microsomes containing ER added after. second sample is in the presence of microsomes containing ER from the start
If the proteins from both samples end up in the ER, then they are inserted after translation, not co-translationally.
If proteins from only the sample containing the microsomes from the start end up in the ER, then they are inserted co-translationally
If no proteins from either sample are found in the ER, then they are never inserted
What does the ER targeting sequence always contain?
A stretch of hydrophobic amino acids
The rest can be variable
N-terminal signal sequence
All secretory proteins (and for many, but not all, proteins co-translationally inserted into the ER) have a cleavable sequence at the N-terminus (the first part of the sequence that’s translated)
It’s the signal that the protein is to be trafficked (co-translatioanlly inserted) into the ER
This sequence is recognized by and binds to SRP as it emerges from the ribosome
How does the N-terminal signal sequence traffick (co-translationally insert) proteins to the ER?
It’s recognized by an SRP (signal recognition particle) as it emerges from the ribosome, stopping translation
SRP, now bound to the ribosome, binds to the SRP receptor on the ER outer membrane. The ribosome is now positioned over the Sec61 translocon in the ER outer membrane
SRP and the SRP-receptor detach from each other
The ribosome resumes translation, but now the polypeptide is being threaded through the Sec61 translocon and into the ER lumen
What quality control mechanism is involved in co-translationally inserting a protein into the ER lumen?
The SRP and SRP-receptor have GTP binding subunits. There is observed GTP hydrolysis upon binding, which acts as quality control
When is Sec61 open?
Only when the ribosome is engaged via SRP and the SRP-receptor
Functions of the Sec61 translocon
Helps polypeptides cross the ER membrane
Enables TMDs to pass sideways through its walls and guides their proper orientation
Binds to and releases ribosomes
Has a tight seal to prevent things (like ATP, Ca2+) from leaking
What tertiary structures make up the Sec61 translocon?
10 alpha-helices
How does Sec61 open and close?
Sec61 is usually plugged + sealed. When unplugged, the peptide chain can pass through its core to the ER lumen. Sec61 can also open laterally in the membrane to allow hydrophobic domains to slip into the lipid bilayer.
Why do all ER signal sequences have a hydrophobic domain?
The hydrophobic portion allows the signal sequence to slip into the lipid bilayer while it’s inside the Sec61 translocon, where it is cleaved from the peptide by a neighboring signal transpeptidase.
Role of BiP (binding protein) in co-translational insertion
BiP is an ATPase in the Hsp70 family. It binds to the signaling complex, then hydrolyzes ATP→ADP+Pi, then in that form it grabs onto the polypeptide. It acts as a clamp to pull the peptide into the lumen and prevents bobbing in the translocon. Molecular ratchet.
Bacteria don’t have ER or Sec61. What analogous structure do they have instead?
SecYEG and SRPs
In the absence of any additional targeting information, where will a water soluble protein in the ER lumen end up?
It will eventually be secreted
Describe the insertion and orientation of Type 1 single pass transmembrane proteins into the ER membrane
The nascent polypeptide is in the translocon, and the N-terminal signal sequence is cleaved
The new N-terminus is now in the ER lumen
A TMD in the polypeptide stops it from going through the translocon; transfer is arrested
The TMD slips through the translocon’s walls and into the lipid bilayer; it is now anchored there
Translation resumes
The remaining C-terminal domain is now in the cytosol with the rest of the fully translated protein
What happens if a nascent polypeptide does NOT have an N-terminal signal sequence?
Secondary rules apply
SRP first recognize an internal hydrophobic sequence (TMD) and brings it to the translocon
The Sec61 looks for positive charges (in the polypeptide) adjacent to the hydrophobic TMD
The side (“side” meaning C-terminal or N-terminal end of the peptide) with adjacent positive charges will be oriented to remain in the cytosol. The side without the adjacent positive charges will be oriented to be in the ER lumen
The hydrophobic TMD slips through the translocon’s walls to be anchored in the lipid bilayer
No cleavage is involved!
A protein with no N-terminal signal sequence is anchored in the ER membrane. It has positively charged amino acids on its N-terminal. Which side of the membrane is the N-terminal on?
Cytosol
A protein with no N-terminal signal sequence is anchored in the ER membrane. It has positively charged amino acids on its N-terminal. Which side of the membrane is the C-terminal on?
Lumen
A protein with no N-terminal signal sequence is anchored in the ER membrane. It has positively charged amino acids on its C-terminal. Which side of the membrane is the N-terminal on?
Lumen
A protein with no N-terminal signal sequence is anchored in the ER membrane. It has positively charged amino acids on its C-terminal. Which side of the membrane is the C-terminal on?
Cytosol
What happens to a polypeptide being co-translationally inserted into the ER without an N-terminal signal sequence and with two TMDs?
Translocon encounters the first hydrophobic TMD and orients it appropriately based on adjacent positive charges
Translation continues
The translocon encounters the second hydrophobic TMD and passes it straight to the lipid bilayer without orienting it
The rest of the peptide is translated in the cytosol
First TMD in a co-translationally inserted protein
Start transfer sequence
Second TMD in a co-translationally inserted protein
Stop transfer sequence
What happens to a polypeptide being co-translationally inserted into the ER without an N-terminal signal sequence and with three or more TMDs?
Follow the same pattern as one with two TMDs
The first TMD provides the orientation
Every TMD after the first is put straight into the membrane without any additional orientation
List two exceptions to the rules of how proteins with TMDs but no N-terminal signal sequence are co-translationally inserted into the ER
Tail anchored proteins and GPI anchored proteins
How are tail anchored proteins co-translationally inserted into the ER?
TMD is at the C-terminal, but it’s too late for SRP recognition. Instead it has its own way to be stuck into the ER membrane. It interacts with a Get3ATPase complex and a distinct translocon. Get3 hydrolyzes two ATP→ADP+Pi to stick the C-terminal hydrophobic TMD in the ER membrane. The rest of the protein remains in the cytosol.
How are GPI anchored proteins co-translationally inserted into the ER?
The protein starts with a cleavable N-terminal SS and a C-terminal TMD. The N-terminal SS is cleaved by the translocon, then the C-terminal TMD anchors it in the membrane. But instead of staying there, the C-terminal TMD is cleaved, and the protein is linked to a GPI anchor
What is the function of the GPI anchor in a GPI anchored protein?
The GPI anchor allows for increased mobility within the membrane.
List 2 key post-translational modifications that occur in the ER lumen
Disulfide bond formation
N-linked glycosylation
How are disulfide bonds formed in the ER lumen?
The ER lumen is an oxidizing environment. The cytoplasm is not. Disulfide bonds can be formed only in the ER lumen because they need an oxidizing environment to form.
Protein Disulfide Isomerase (PDI) exists within the ER lumen. It helps mix + match amino acids that form disulfide bonds, and transfers oxidizing equivalents to substrate proteins
PDI assists with both making and rearranging the disulfide bonds
For transmembrane proteins, the oxidation happens only in the exoplasmic side
What is Protein Disulfide Isomerase (PDI) and what does it do?
Protein Disulfide Isomerase (PDI) exists within the ER lumen. It helps mix + match amino acids that form disulfide bonds, and transfers oxidizing equivalents to substrate proteins
PDI assists with both making and rearranging the disulfide bonds
Where does disulfide bond formation happen?
Only in the ER lumen, never in the cytoplasm!
What does the “N” mean in N-linked glycosylation?
“N” means asparagine, not the N-terminus
Funnily enough, the asparagine can’t be in the protein’s N-terminal end. It has to be in the C-terminal end
How does N-linked glycosylation occur?
In the ER lumen, oligosaccharyl transferase takes a premade dolichol (a lipid linked oligosaccharide) and a protein with an N-glycosylation site and links the dolichol’s oligosaccharide to an asparagine within the protein
Can N-linked glycosylation occur on an asparagine in a protein’s C-terminal end?
Yes
Can N-linked glycosylation occur on an asparagine in a protein’s N-terminal end?
No
What form(s) of energy are used to create the dolichols for N-linked glycosylation?
GTP and UTP
Notably no ATP is involved
Which leaflet/membrane side is sphingomyelin predominantly found on?
Exterior
Which leaflet/membrane side are glycolipids predominantly found on?
Exterior
Which leaflet/membrane side is phosphatidylcholine predominantly found on?
Exterior
Which leaflet/membrane side is cholesterol predominantly found on?
Both leaflets
Which leaflet/membrane side is phosphatidylserine predominantly found on?
Cytosol
Unless during apoptosis, then which it’s flipped to the exterior leaflet
Which leaflet/membrane side is phosphatidylinositol predominantly found on?
Cytosol
Which leaflet/membrane side is phosphatidylethanolamine predominantly found on?
Cytosol
What is the functional relevance of phospholipid asymmetries in a plasma membrane?
Glycolipids on the exoplasmic/exterior side like PI and PS are core to normal cell signaling. Exoplasmic PS signals to other cells that its in apoptosis and that nearby cells need to eat the dying guy.
What side of the membrane are the enzymes involved in synthesizing dolichol found?
Cytosolic side
As synthesis enzymes add new lipids to the cytosolic side of the membrane, the two leaflets will eventually become unbalanced (too many lipids on the cytosolic side). How is this solved?
Scramblases
Scramblases are membrane proteins that even the distribution of newly synthesized phospholipids in the smooth ER membrane. They shield the movement of the phospholipid heads, allowing the excess lipids to flip to the other side. They move the lipids down a gradient, so no ATP required.
What side of the membrane are the enzymes involved in synthesizing lipids found?
Cytosolic side of the smooth ER membrane
What proteins do the work of scramblases in the plasma membrane?
Flippases
However, flippase moves lipids UP a gradient, so they do require ATP. They use a P-type ATPase pump for their ATP hydrolysis
How do flippases handle large substrates?
Credit card model
Only the head group of the phospholipid goes through the flippase’s protected interior space to be flipped to the other side
Where is phosphatidylserine located during apoptosis?
The exoplasmic leaflet
It’s the signal to other cells that it’s dying and needs to be eaten
What protein flips phosphatidylserine to the exoplasmic leaflet during apoptosis?
Scramblase
