Organelles And Protein Sorting Flashcards
(103 cards)
1
Q
What makes up the volume of a cell?
A
Half cytosol, half membrane
2
Q
Where is the mostmembrane found in the cell?
A
The ER, so whatever cells have more ER ( to synthesize proteins) have more membrane
-the plasma membrane only makes up small fraction of
3
Q
What are the steps in the evolution of Eukaryotes? (Endosymbiotic theory)
A
1.) loss of rigid cell wall in ancient anaerobic archon caused horizontal gene transfers to come into existence.
2.) ivagi of other prokaryote occur, speeding, evolutionary processes.
2.) membranes, increasingly enclosed the chromosome of anaerobic arc help protect it.
4.) development of multiple mitochondria provides energy for the evolution of additional systems and much larger cells
4
Q
What is the Nucleolus?
A
Condensed region in the cell where ribosomes are formed (inside the nucleus)
5
Q
Peroxisomes?
A
Metabolize waste in the cytoplasm of the cell
6
Q
Endoplasmic reticulum (rough and smooth portions)
A
Rough: associated with ribosomes, externally. Makes secretary and membrane proteins
Smooth : makes lipids, lack of external ribosomes
7
Q
Microtubules?
A
Form the mitotic spindle and maintain cell shape
8
Q
Centrosome?
A
Microtubule-organizing center
9
Q
Intermediate filaments?
A
Fibrous proteins that hold organelles in place
10
Q
Lysosome?
A
Digest cellular food
11
Q
Golgi apparatus?
A
Modifies proteins
12
Q
The first eukaryotic cells were ….?
A
Aerobic
13
Q
What did eukaryotic cells evolve from?
A
Anaerobic archaeon, which joined forces with aerobic bacteria
14
Q
What does it mean if two compartments are topologically equivalent?
A
You do not need to go through a membrane to get from one department to the other, you can go through “pores”
-Identical environments
15
Q
What parts of the cell are topologically, equivalent, and shown as “gray”?
A
The nucleus and the cytoplasm
16
Q
What compartments in the cell are topologically, equivalent, and shown as “pink”?
A
The ER, the Golgi apparatus, mitochondria, endosomes, lysosomes, secretory vessels, nuclear envelope
17
Q
What is special about the lumen of many organelles?
A
It is topologically equivalent with the extra cellular space
18
Q
What is vesicular transport?
A
When a molecule is transported as a vesicle, it has a protective bubble which fuses to a membrane and goes through the membrane in that bubble
-doesn’t need to pass through a membrane because going from pink to pink, instead is transported through it in a safety bubble (which keeps inside continents safe from external environment )
19
Q
What is transmembrane transport/protein translocation?
A
A unidirectional method of transport from the cytosol to either: the mitochondria, the endoplasmic reticulum, peroxisomes.
-Goes between two topologically different organelles, so needs to pass through a membrane.
20
Q
Where does vesicular transport occur?
A
From the Golgi to out of the cell
21
Q
What is gated transport?
A
Only occurs between the cytosol and the nucleus, goes through pores because both are topologically equivalent
22
Q
What are the three main transportation systems to move proteins in the cell?
A
Gated transport, transmembrane transport, vesicular transport
23
Q
What occurs during vesicular transport?
A
-Donor compartment causes a vesicle to “bud off”, to carry cargo.
-Maintains polarity inside outside stays outside.
-Surrounded by a membrane (outer leaflet and inner leaflet)
-cargo fuses to the target compartment (contents unchanged)
-The membrane of the vesicle becomes the membrane of the target
24
Q
What are the six signal sequences that direct protein traffic?
A
1: no signal= cytoplasm (default)
2: nuclear localization signal (NLS)
3: nuclear export signal (NES)
4: import into ER
5: retention into ER
6: import into mitochondria
25
What is the nuclear localization signal (NLS)?
-can be anywhere in the protein
-Signified by positively charged amino acids (specifically lysine and arginine) (K and R)
-Protein is made cytoplasm. The signal is made/read, protein is then sent to nucleus.
26
What is the nuclear export signal (NES)?
-can be anywhere on the protein
-Follows a 3,2,1Lucine pattern
(L-X-X-X-L-X-X-L-X-L) (X= any amino acid)
-Goes to nucleus does job and leaves (lots of back-and-forth)
27
What is the import into ER signal?
-found on the N-terminal
-Signified by 5 to 10 hydrophobic amino acids (can be any)
28
What is the retention into ER signal?
-Found on the C- terminal
- signified with KDEL (LYS-ASP-GLU-LEU) chain
-Not sufficient in itself, will keep protein in the ER, but must also have import sequence to get to ER or otherwise useless
29
What is the import into mitochondria signal?
-found on the N-terminal
-Alternating positive and hydrophobic amino acids
30
What happens if just one amino acid is mutated on the NLS?
It will disrupt nuclear localization, will not work in protein will stay in the cytosol
31
What is special about the endoplasmic reticulum membrane?
It is continuous with the nuclear membrane (things found in the Perinuclear space inside the outer and inner membrane will also be found in the ER)
32
How many membranes does the nucleus have?
Two, the inner and outer nuclear membrane
-A double layered envelope that surrounds the nucleus
-periNuclear space is inbetween the two membranes
33
What is the nuclear lamina?
A protein mesh work that provides structural support and anchors chromatin
34
What are nuclear pore complexes (NPC’s)?
“-nucleus gate keepers”
-3 to 4000 NPC per nucleus, each composed of 30 nuclearporins (NUPs)
35
What are cytosolic fibrils?
-they look like oily spaghetti
-Have a high affinity for FG repeats (phenylalanine, glycine) which help recruit NLS signal
-Helps NLS signals proteins, get into the nucleus 
36
At what size is a molecule considered “small” and able to pass through the membrane freely?
60 kDa or lower
37
What is an importin/nuclear import receptor?
-bind to NLS of proteins
-Facilitates docking NLS protein at a nuclear poor complex in order to gain entry into the nucleus
-Multiple shapes/kinds
38
What role does cytosolic nuclear pore fibrils play in transporting the protein to NPC?
Affinity for FG repeats “dock” the NLS/transport receptor onto the NPC
39
What is Ran?
A GTP-ase, it can exist as ran GDP or ran GTP
-a molecular “switch”, very similar to ATP
40
What is ran-GAP?
( GTPase activating protein)
-enzyme that works in the CYTOSOL that triggers conversion of ran-GTP to ran-GDP
-creates high conc of GDP in the cytosol
41
What is ran-GEF?
(GTPase Exchange Factor)
-an enzyme in the nucleus that converts Ran-GDP to Ran-GTP
-is stuck to chromatin inside the nucleus
-causes high concentration of Ran-GTP in the nucleus
42
What are the steps to getting a protein with the NLS signal into the nucleus, and the cycle of Ran-GTP and GDP?
1: nuclear transport receptor (importin) and cargo protein is attracted by FG repeats to cytosolic nuclear pore fibrils
2: goes through the nuclear poor complex
3: Receptor transports cargo protein into the nucleus
4: ran GTP binds to important and cargo protein is therefore released
5: cargo protein is now delivered to be free in the nucleus
6: Importin (which is now bound to Ran-GTP) is exported back to the cytoplasm, where GTP will be hydrolyzed to GDP, lose its affinity for importin, and they will go their separate ways
7: GDP, now dissociated, will be transported via (NTF2) transporter back into the nucleus to be used again
43
What is ran-GDP?
-Found in the Cytosol
-Transported back into the nucleus by NTF2 transporter (to be made into GTP by Ran-GEF)
-Has no affinity for importin or exportin (releases both of these into the side of all)
44
What is ran-GTP?
-found in the nucleus
-Binds to importin, releasing cargo protein into the nucleus
-Has high affinity for importin
- gets exported back to the cytoplasm after with importin (where it will be hydrolyzed by ran-GAP which expels a Pi)
In export:
-binds to exportin and cargo protein, leaves the nucleus with both of them
-loses affinity and dissociates once hydrolyzed
45
What are “piggyback” proteins?
-proteins that do not have an NLS signal of their own but bind to a protein with NLS in order to get allowed into the nucleus
-ex: Actin (microfilaments) do this by associating with cofilin (actin depolymerizing protein)
-in some cases, these piggyback proteins may actually block the NLS to prevent nuclear entry! (Via phosphorylation)
46
Where does the mitochondria get most of its proteins?
Via the nucleus from importing
-odd because it contains its own DNA, ribosomes, and protein production components, but is dependent for importation of proteins
47
What is the structure of the mitochondria?
There is an OUTER membrane and an INNER membrane, which surrounds the MATRIX SPACE which is the true mitochondria inside
-The space in between the outer and inner membrane is called the INTERmembrane space
-The space in between the wiggles of the matrix is called the CRISTAE space
( we need to be able to transport through all the layers )
48
What are the four different complexes involved with mitochondrial transport?
1: the TOM complex (translocase of outer membrane)
2: the TIM complex (translocase of inner membrane)
3: the SAM complex (sorting and assembly machinery) (outer membrane)
4: the OXA complex (cytochrome oxidize activity) (inner membrane)
49
What is the definition of a translocase?
A complex embedded in one of the mitochondrial membranes to thread unfolded proteins through
50
In what condition do proteins enter the mitochondria?
-unfolded, the opposite of nuclear transport
-proteins are synthesized in the cytosol but not folded until in the mitochondrial matrix
51
What is the order of events required to get a protein into the mitochondrial matrix?
1: the mitochondrial import sequence binds to receptor protein in TOM complex
2:TOM THEN THREADS IMPORT PROTEIN THROUGH THE OUTER-MEMBRANE, UNFOLDED, AND FEEDS INTO THE TIM COMPLEX
3: TIM then performs translocation of the protein into the matrix
4: the import sequence is then cleaved by Signal Peptidase off the protein
5: the mature mitochondrial protein is then folded in the matrix space
52
Where do chaperones play a role in mitochondrial transport?
-in the cytosol, the import protein is covered with cytosolic Hsp70 chaperones
-As it gets fed to the TOM complex, ATP is required to strip off the chaperones and push the protein in
-(1 ATP per chaperone per taking off/putting on- a.k.a. expensive)
-once in the matrix the mitochondrial Hsp70 chaperones are then stuck back on to make it fold properly (costs the same amount of ATP as when protein entered)
53
What energetics help fuel mitochondrial transport?
-The matrix of the mitochondria has a negative membrane potential because of the electron transport chain, this helps pull positively charged signal sequence through
-Electrochemical gradient (negative innner membrane charge) helps pull protein through TOM and TIM
54
What are the two ways to make inner membrane transmembrane proteins in the mitochondria?
1:TIM complex: protein is never released into matrix, bc there is a hydrophobic Stop-Transfer sequence immediately behind the signal sequence.
- that stops transport into matrix and send it to become an inner membrane protein
2:OXA complex: protein is allowed into the matrix, separate second signal sequence then sends it to the OXA complex
-Can make inner membrane transmembrane proteins out of both mitochondrial proteins or imported proteins
55
How do you make outer membrane transmembrane proteins on the mitochondria?
-Signal tells TOM not to send to TIM, instead, the protein gets covered in chaperones and sent to SAM complex
-here it becomes folded outer membrane protein
-for example, outer mitochondrial porins
56
In cells that make proteins, what will be found in a higher amount in the cell?
Rough endoplasmic reticulum (because makes proteins)
The cells will also therefore have more membrane, due to the high membrane percentage that is present in the ER
57
How is ER transport different from nuclei and mitochondrial transport?
ER importation is done as: “ co-translational translocation”
(meaning that translation is occurring as it is imported)
-Different from other transport, because in nucleus and mitochondria, translocation occurs after translation is complete
58
Why does the rough ER have ribosomes attached to its membrane?
Because as proteins are being imported through the membrane, the ribosomes are anchored there after translation begins by sticking to the mRNA
59
What is the SRP?
The signal recognition particle
-Wraps around large ribosome as soon as signal peptide has been translated
-Pauses translation until SRP binds to SRP receptor (an integral Transmembrane ER protein)
60
What happens once SRP binds to the SRP receptor? (final steps of protein and importation into ER)
The SRP-ribosome is brought to a Sec61 protein translocator (separate from the SRP receptor)
-The protein is then fed through Sec61 into the lumen of the ER
-The signal sequence is stuck to the Sec61 “ tunnel” at this point, and the rest of the protein is fed through Sec61 unfolded, and as a fish hook/U shape
-Signal peptidase then comes in cleaves off, initial signal sequence on the N terminal
-Protein now folds in ER lumen and job is done
61
Why does Sec61 need to be closed when not actively importing proteins into the ER?
So that calcium cannot leak out (there is lots of calcium inside)
62
What is special about transmembrane proteins that are made in the ER membrane?
Because the lumen of the ER is topologically equivalent, with the extracellular space, proteins for external side of plasma membrane are made in the ER
63
What is the difference between the stop transfer sequence and the start transfer sequence for importing into the ER?
Start transfer sequence: the first signal sequence of 5 to 10 hydrophobic amino acids
-Can be on the N-terminal (in which it will always be cleaved at the last step) or an internal signal sequence
-Signifies the start of importation into the ER lumen via Sec61
Stop transfer sequence: the second signal sequence of 5 to 10 hydrophobic amino acids
-Always internal (never cleaved)
-Always becomes transmembrane segment
* the status of start or stop depends on the order. They are on the protein.
64
What determines the amount of transmembrane segments in a transmembrane protein in the ER/plasma membrane?
The number of internal signal sequences (5 to 10 hydrophobic amino acids)
65
How can you tell how many transmembrane segments are on a protein?
Hydropathy index/plot!
-The amount of “peaks”
-SRP recognizes the first hydrophobic segment to emerge from the ribosome to set the “ reading frame”
-Subsequent hydrophobic segments are recognized by Sec61
66
When a transmembrane protein is passed from the ER to the plasma membrane, what side of the membrane will go where?
The cytosolic facing side will still face the cytoplasm (positive charge on this side)
The ER lumen side will become the outside exterior of the cell (never positive on this side)
67
What happens once Sec61 identifies an internal signal sequence?
Sec61 then opens a “ back door” that passes the internal signal sequence into the membrane and that has now become a transmembrane protein
-Sec61 recognizes any subsequent internal signal sequences to create as additional transmembrane passes
68
How does the charge of an internal signal sequence affect its becoming a transmembrane protein in the ER?
If on one side of the internal signal sequence, there is a positive charge that side must always be on the cytosol side of the membrane , and will never be allowed into the ER lumen
-“ positive charges on proteins will always face the cytosol”
69
If there is an internal signal sequence for the ER as the start signal in a transmembrane protein, what side will the n-terminus be on?
The cytosolic side (internal signal is like fishhook/U)
*Assuming there is no charges
70
If there is an odd amount of internal signal sequences into the ER, where will N and C terminal be in relation to each other?
On opposite sides of the membrane
-vise verse for even
71
If there is an n-terminal signal sequence on a transmembrane protein into the ER, what side of the membrane will N terminal be on?
In ER lumen (bc rest of protein gets anchored there after signal cleared by signal peptidase)
72
Where are sugars added to proteins that will be sent to the plasma membrane?
In the ER, this is called being “Glycosylated”
73
What does oligosaccharyl transferase do?
-recognizes asparagine + signal on protein, and attaches oligosaccharide tree to asparagine side chain at the NH site of that protein
74
How many eukaryotic proteins are glycosylated?
Half of all of them!
75
What is an oligosaccharde tree made of?
Glucose, mannose, and n-acetylglucosamine
- this attaches to NH of asparagine side chain
76
What is the Dolichol?
The lipid anchor for the oligosaccharide
- where oligotransferase grabs sugars from to add to ASN
77
When is the oligosaccharide added to a protein?
During translocation into the ER membrane (as it is threaded through the translocation channel)
78
Why is it odd that the glycosylated process starts inside the cytoplasm?
-because it is energy expensive to move the half formed sugar tree in through the membrane
-“The sugars are first activated in the side of plasm by formation of nucleotide (UDP, or GDP) sugar intermediates, which then donate their sugar to the lipid in an orderly sequence”
79
Why do we need the oligosaccharide tree?
- it serves as a timing mechanism in the ER
-it tells how long a protein has been in the ER because glycosidases are constantly trimming away glucose as they fold
80
What happens once the protein reaches its final glucose when folding in the ER?
-It sounds the alarm and calls in chaperone proteins to help it fold
81
What happens once a protein reaches its final (fourth) Mannose while trying to fold in the ER?
The protein is retrotranslocated and destroyed, because the Mannosidases have been chewing away the mannoses for too long. Meaning, there’s something wrong with this protein.
82
What chaperoned proteins are called in to help proteins fold in the ER once the final glucose has been reached?
Calnexin: ER membrane bound chaperone protein
Calreticulin: same function as Calnexin, but for soluble proteins (not anchored)
BiP and ERp57: ER chaperones that bind to unfolded or misfolded proteins, and prevent their exit from the ER “ catch proteins and prevent aggregation and escape”
83
Where is a protein sent once it is properly folded in the ER?
The Golgi apparatus!
84
What happens after a final glucose was added, chaperones were added, and yet the protein is still unfolded in the ER?
Another glucose is added by glucosyl transferase and calnexin is added again for another attempt
- keeps going until final mannose is reached (3?)
85
How is protein freed from calnexin?
Glucosidase trims off final glucose
-then, BiP checks it to approve or fail
86
What occurs during retrotranslocation and destruction of unfolded proteins?
1: lectin binds to oligisaccharide tree, and brings it to translocator complex
2: protein translocated complex is powered by AAA Atpase, to pull the protein back into the cytosol
3: polyubiquitin chain add ubiquitin to the protein, which will then be destroyed by a proteasome
87
What happens when excessive misfolded proteins start to file up in the ER?
- The unfolded protein response (UPR) is activated, and produces more chaperone proteins
-ER size expands as chaperone proteins are imported
88
On what side of the ER membrane does phospholipid synthesis occur? Why is this a problem?
-The cytosolic side (it is catalyzed by enzymes here)
-we only make membrane on the side of smooth ER, so only cytosolic side grows when we make membrane
89
How is the ER membrane asymmetry fixed?
-Scramblases!
-relies on scramblase to even out membrane so that there is an equal amount on both sides
-However, is dangerous, because could flip out phosphatidylserine
90
How do we fix the work of scramblases on our membrane?
-we rely on flippases!
-they make sure phosphatidylserine stays on inside of membrane when scramblase scrambles!
91
What does the SRP bind to first?
The large ribosomal subunit
92
Who adds a final glucose to a protein after a failed folding attempt?
Glucosyl transferase!
93
What is the stop and send to Tim sequence in mitochondrial transport after the transport signal?
Hydrophobic stop transfer sequence!
94
Do chaperone proteins (HSP70) exist in intermembrane space of mitochondria?
Yez
95
When is the signal sequence cleaved during import to ER?
During its translocation! While the protein is being fed through SEC61
-ergo is N terminus and no charges, the N terminal will be released into ER lumen
96
If there’s an internal start, where will the N terminus be?
In the cytosol! Unless there are charges present
97
If there is an N-terminus signal sequence for ER and no charges, which side of the membrane will the N-terminus be?
The ER lumen side/outside the cell!
98
What determines the orientation of SINGLE pass transmembrane proteins?
Nearby amino acids! (Ex: charges)
99
What determines orientation of MULTI pass transmembrane proteins?
If internal start: the N-terminus will stay in the cytosol
(Unless there is positive charge!)
100
Where are scramblease and flippase located?
Scramblease: the smooth ER membrane
Flippase: the PM
101
What is the signal that a protein has been trying to fold in the ER for too long and needs to be retrotrabslocated/destroyed?
A critical mannose is chewed off!
102
What strips off the olgisaccharide chains?
N-glycanase!
103
What straightens a misfolded protein before degredation?
Disulfide isomerase!
