Midterm 2 Flashcards
1
Q
Protein Targeting: A fundamental cellular process
A
Proteins that have a specific destination contain a sorting signal in their sequence in amino acid encoded originally in the DNA
2
Q
macromolecules contain within their sequence all the information that governs their processing and transport; examples:
A
eg. promoter sites on DNA, intron and exon processing/splicing, histone modification (methylation, phosphorylation)for chromatin remodelling, targeting signals (e.g. NLS), information for 5’-cap and 3’-poly A tail, targeting to all organelles, targeting to membranes, proper folding and the final 3D conformation
3
Q
Proteins with no sorting signals remain in the…..
A
cytosol
4
Q
All translated in the ___ from a pool of ribosomes and the ________determine its final destination
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cytoplasm, signal sequences
5
Q
Sorting signals:
A
Direct the protein to a specific organelle
Must be present for protein to leave the cytosol compartment
6
Q
KDEL
A
KDEL is the signal that signals retention in the lumen of the ER
Likely contain chain of hydrophobic amino acids
ER(resident)
post-translational
mixed properties
not cleaved
7
Q
Post-translational trafficking of proteins
A
Not targeted to their destination until fully translated in the cytosol
Signal recognized once fully translated
Ribosomes remain “free” in the cytosol
Membrane-bound free ribosomes which are structurally and functionally identical, differ only in the proteins that they are making at a particular time
Completed polypeptide goes to its functional destination depending on its sorting signal
Polypeptide may be folded or unfolded
Nuclear proteins folded
Passed through translocators of Mitochondrial and chloroplast proteins (encoded by nuclear genes)- unfolded in order to enter organelles
Proteins that are translated through nuclear pores
8
Q
Co-translational trafficking of proteins
A
Proteins that enter the endomembrane system
Ribosome starts translating, and as done translating targeting signal, the targeting signal is recognized and is brought to ER membrane system for insertion
Ribosomes attach to ER membrane
Protein is threaded through ER membrane as its being translated
Proteins either stay in ER, or continue to other compartments of the Endomembrane system
Synthesis is always initiated in the cytosol
9
Q
ER/Golgi/lysosomal proteins; 2 types:
A
Secreted proteins (secretory proteins)- proteins that are secreted out of the cell Membrane proteins- proteins that are inserted into the membranes of the ER
10
Q
Endomembrane System
A
membrane-bound compartments involved in processing and movement of proteins and membranes
11
Q
what determines whether ribosomes are attached to the ER
A
Whether a ribosome becomes attached to the ER depends on the mRNA being translated
12
Q
RER and SER
A
RER are sites of secretory protein synthesis
SER are site of lipid and steroid synthesis
RER have ribosomes docked onto them while SER don’t
ER is the starting point for protein traveling the endomembrane system
Rough ER and smooth ER are continuous
RER has ribosomes docked onto them but SER does not
13
Q
proteins are made by
A
Common pool of ribosomes for cytosolic proteins and proteins destined for membrane bound compartments including the ER
14
Q
Functions of ER:
A
Entry point for proteins into the secretory pathway post-translational modifications protein folding by chaperone proteins Quality control site membrane lipid biosynthesis on SER Controls calcium levels in cytoplasm
15
Q
Entry point for proteins into the secretory pathway
A
Entry point for proteins into the secretory pathway (co translational transfer across RER membrane then transported by vesicular traffic to Golgi, etc.)- entry for endomembrane system
16
Q
post-translational modifications of ER
A
Site of post-translational modifications of proteins that enter the endomembrane network, eg protein disulfide isomerase forms disulfide bonds here, glycosylation starts here
17
Q
protein folding by chaperone proteins
A
Site of protein folding by chaperone proteins, such as BiP (binding protein) which prevent hydrophobic domains of proteins from aggregating and promotes proper folding
18
Q
Quality control site ER
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Quality control site- check for defective proteins (proteins are exported for degradation from ER if they are not properly assembled)
19
Q
membrane lipid biosynthesis on SER
A
Site of membrane lipid biosynthesis on SER (sterols and glycerolipids). Cells producing large amounts of lipids have abundant SER
20
Q
New phospholipids are synthesized by the enzymes in the cytoplasmic face of the ER
A
Inserted into the cytosolic leaflet of the membrane
Flippase will flip the phospholipid to the ER -Asymmetry is established by Flippase enzymes
Different chemicals on each leaflet allows us to know which is the cytosolic side and which is the non-cytosolic face
lumen side to balance out the lipid bilayer
Membranes get sent along the endomembrane system and reaches the other organelles
Subject to modifications
21
Q
Types of proteins targeted to the ER
A
- Soluble proteins destined for secretion
- Membrane proteins that are inserted into the ER membrane co-translationally- maybe destined anywhere in the EM system
- Resident proteins of the endomembrane system (can be soluble or membrane proteins)
ER resident proteins (e.g. chaperone proteins)
Golgi resident proteins
Lysosomal resident enzymes
22
Q
Targeting to ER:
A
Signal encoded within the protein
Receptor (signal recognition particle, SRP) that recognizes and binds the signal
23
Q
The ER signal sequence is a ____
A
chain of nonpolar uncharged amino acids
24
Q
The ER signal sequence is ________ for entering the ER - _________experiment
The ER signal sequence is for entering the ER - _________ experiment
A
necessary (required), loss of function, sufficient (enough), gain of function
25
ER signal sequence and signal recognition particles (SRP) direct ribosome to ER membrane steps:
Ribosome and mRNA bind and starts to translate
1. SRP binds to the exposed ER signal sequence while being translated(a.k.a “Start Transfer Sequence”) and to the ribosome (slowing protein synthesis) Note: the signal shown here is an N-terminal ER signal sequence
2. SRP and ribosome is complex then binds to SRP receptor in the ER membrane and brings it close to protein translocator
3. Protein translocation channel assembles and inserts the polypeptide chain (signal sequence) into the membrane and it remains embedded in the membrane and starts its transfer across the bilayer. Rest of the polypeptide is threaded through the protein translocator as it is being translated
If there is no other signal, the C-terminal signal goes all the way through
26
If the protein is soluble:
A soluble protein crosses the ER membrane into the lumen- has ONLY the N-terminal Signal Sequence
1. Protein translocation channel binds the N-terminal Signal Sequence and actively transfers the polypeptide across the bilayer
2. The N-terminal Signal Sequence is cleaved from the growing protein by a Signal Peptidase
3. The translocated protein is released as a soluble protein into the ER lumen if there are no other signal sequence (no other transmembrane region)
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Start transfer sequences
N-terminal signal sequence (N-terminal start transfer sequence)- right at the N-terminal end
Very first signal sequence encountered will bring the ribosome to the ER membrane and direct translocation
Initiates transfer of protein across the ER membrane
Cleaved off
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In terms of transmembrane proteins
Nothing is cleaved; region is embedded in the membrane
Location of signal sequence that determines the orientation of transmembrane protein
Transmembrane proteins with the N-terminal facing the ER lumen
Transmembrane proteins with the N-terminal facing the cytosol
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N-terminal sequence signal sequence and an internal Stop-Transfer Sequence are involved in integration of one-pass transmembrane protein
its N-terminus in the ER lumen
As soon as the stop transfer signal is recognized, the ribosome lets go and no longer feeds polypeptide in
Signal peptidase cuts n-terminal sequence off and the N terminal end ends up in the ER lumen while the C-terminal end is left in the cytosolic side.
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a pair of start transfer (internal sequence, not N-terminal) and stop transfer sequences generate double-pass through the membrane (both N- and C- terminus in the cytosol)
Internal start transfer sequence
Initiates transfer of protein across ER membrane
Everything between the start and the stop sequence are fed through
Is a membrane crossing domain
Not cleaved off because the N-terminal sequence is no longer at the N-terminus
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Stop transfer sequence
Stop transfer of protein across ER membrane
| Is a membrane crossing domain
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The first transfer sequence will be a “start transfer” sequence
This causes the polypeptide to feed into the ER after this “start” sequence
If this is right at the N-terminal end, it will be cleaved after the protein synthesis
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The second transfer sequence will be a “stop transfer” sequence
This causes the polypeptide to stop entering the ER after this “stop” sequence
These “start” and “stop” sequences alternate when several are found within a polypeptide
34
Summary of protein insertion into membranes
Proteins can be inserted in any orientation depending on the occurrence and placing of signal peptide, Start, and stop transfer sequences
Both the start and stop transfer sequences are membrane crossing domains
The N-terminal sequences is always cleaved, the others always remain
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Additional rules for targeting signals and membrane integration
If the N-terminal start transfer sequence (signal sequence) is present the N-terminal end of the protein will always be in the ER lumen
If the last membrane crossing domain is a stop transfer sequence, the C-terminal end of the protein will be in the cytosol
If the last membrane crossing domain is a start transfer sequence, the C-terminal end of the protein will be in the ER lumen
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The presence of a N-terminal signal sequence determines if a protein in the endomembrane system is ...
the membrane protein or free in the lumen as the N-terminal sequence allows the polypeptide to enter the membrane.
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Proteins expected to be targeted to the ER and,
| Proteins are processed:
Folded
Glycosylated
Modified
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All proteins are processed once they are translated on ribosomes; In the cytosol (cytosolic proteins)
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Folding
Covalent modification (phosphorylation, acetylation, methylation)
Cleavage- proteins are cut into pieces
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39
All proteins are processed once they are translated on ribosomes
In the ER/Golgi system (proteins of the endomembrane system)
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Folding
Covalent modification
Cleavage
Glycosylation
Disulfide bond formation
Disulfide bond formation is rare in cytosolic environment as it is too much of a reducing environment
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Carbohydrate modifications begin in the ER
Carbohydrates are only attached on the non-cytosolic side of the plasma membrane, always
This orientation begins in the ER and is maintained throughout the endomembrane system
Glycocalyx: the “sugar coat” on the plasma membrane
41
Early glycosylation of proteins in the ER:
1. Oligosaccharides are added to dolichol (phospholipids) anchored in the cytosolic face
2. A flippase enzyme moves this structure into the ER lumen
3. This oligosaccharide structure is transferred from dolichol to the growing polypeptide.
The transferase recognizes “Asn-X” sequences (where “X” is Ser or Thr) and links the oligosaccharide to Asn
42
Early glycosylation of proteins in the ER Summary:
Glycolipid units are:
Initially synthesized in the cytosol and embedded in the cytosolic face of the membrane, they are then
Flipped to the lumen side of the membrane by a flippase enzyme
Joined covalently to Asn in an Asn-X (Ser or Thr) sequence tag
The oligosaccharides are added as an intact prefabricated unit, consisted of 14 linked sugar residues transferred from phospholipid anchor (dolichol) in the membrane
The enzymes that transfer the oligosaccharides are located in the lumen of the ER
These oligosaccharides will be processed further in the Golgi
43
Targeting to ER: protein folding
| Newly synthesized proteins are at risk of misfolding and clumping
H-bond could H-bond with other molecules instead of within itself and result in tangles
H-bonds with water and cause tangles until it likely finds correct H-bond partner
Hydrophobic areas tend to clump together in aqueous solution and are less likely to exchange, leading to clumps
Polypeptide may misfold- form interactions other than the ones necessary for proper structure and function
Interactions may form between several new polypeptides, forming clumps
44
Chaperones
Misfolded proteins in the ER lumen trigger the production of Chaperones (=feedback loop)
Chaperone (must contain signal sequence if it needs to enter ER) proteins interact with polypeptides and can unfold and refold them
45
Targeting to ER: chaperone proteins
1. Aid in the proper folding of the proteins
2. Prevent misfolded or partially assembled proteins from leaving ER
Any misfolded proteins that cannot be refolded are sent back into the cytosol for degradation
46
ubiquitin
Misfolded protein tagged with ubiquitin molecules in the cytosol
Ubiquitin-tagged proteins recognized and degraded by proteasome (recognizes ubiquitin tag)
Misfolded proteins are not properly folded are destroyed
In the ER, improperly folded proteins are returned to the cytosol and destroyed in the same way as are improperly folded
47
Endomembrane system
ER, golgi, Late endosome, lysosome, early endosome, cell exterior, secretory vesicles
48
Endomembrane system: All of these pathways use vesicular transport
Includes:
Secretory pathway
Lysosomal pathway
Endocytic pathway
49
Secretory:
all the proteins going through the endomembrane system head out to the surface to the PM or secreted out of the cell, or diverted to the lysosome
50
Endocytic
materials are taken in from the outside and are diverted to the lysosome
51
Recycling and retrieval
all the ER and golgi resident proteins: they sometimes get caught up in the transport vesicles into the wrong compartment; they need this system to send them back
52
Principle process throughout the endomembrane system:
vesicles bud from a donor compartment and fuse with a target compartment
The soluble cargo gets released into the lumen of the target compartment and transmembrane proteins are now in the membrane of the target compartment
53
Vesicles must take the correct cargo from the donor compartment to the correct target compartment
This means that...
1. Correct cargo MUST get into the correct vesicle
2. The correct vesicle MUST get to the correct destination
This is controlled by steps involved in vesicle formation, transport, and docking.
54
4 stages of vesicle transport
1. formation
2. Transport- happens in co-action with cytoskeleton
4. Docking
5. Fusion
(resetting the system)
55
Formation steps
Step 1: Budding
Transported protein is recognized/bound by a receptor protein in the membrane and these receptors concentrate cargo on one location in the membrane
Adaptor proteins recognize and bind to the cytosolic portion of the receptor
Coat proteins bind to the adaptor proteins- because of shape of coat protein, it helps to bend and curve it off, causing the budding process driven by GTP
regulated by a small GTP-binding protein (GTPase)
Bound to GTP: active - allows adaptors and coat proteins to bind
Step 2: Pinching off
Coat proteins bend the membrane into vesicle
The vesicle pinches off from the donor membrane (sometimes with help from a scission protein)
Once it pinches up, we have a vesicle
Step 3: Coat is shed
GTP hydrolyzes to GDP: inactive- GTPases, coat proteins, and adaptor dissociate from vesicle
Step 4: Naked vesicle ready to fuse
56
Non-cytosolic side:
Faces lumen or outside the cell
Soluble cargo accumulates and binds a cargo receptor in the donor membrane
Membrane cargo often interact directly with adaptors on their cytosolic domain
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Cytosolic side:
Cargo receptor or membrane cargo interact with coat adaptor molecules
Adapter molecules interact with coat proteins
Membrane budding out
Cargo is concentrated on cytosolic side of membrane
58
Different types of coat proteins (and associated proteins) used in vesicle formation in different parts of the endomembrane system.
Correct cargo makes it into correct vesicles by receptor proteins and coat proteins
Coat proteins work with the cargo receptor
Clathrin works between the trans golgi network and the plasma membrane; works in secretion and endocytosis
COPII mediate transport from the ER to the golgi
COPI coats mediate transport material from the golgi to the ER
59
Clathrin-coated vesicles
Primarily involved in endocytosis, traffic to lysosome (golgi to the cell membrane) & receptor recycling
Dynamin required for budding- to pinch off the vesicles
Cargo: cholesterol, hormones, old protein receptor and other old proteines (recycled)
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mutation in dynamin GTPase:
Dynamin and associated proteins
| Blocked by some dynamin mutations
61
COPII-coated vesicles
Primarily involved in ER -> golgi traffic (going forward)
Cargo, cargo receptor that binds to soluble cargo proteins or transmembrane protein that are acting as cargo which are bound by adaptor proteins- regulated by small GTP binding proteins
COPII proteins that recognize the adaptors and help bend the vesicle into shape
Dynamin-like protein not required to pinch off
Moving forward: anterograde
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COPI- coated vesicles
Primarily involved in golgi -> ER traffic
Dynamin-like protein not required to pinch up
KDEL (ER retention signal)- soluble ER membrane proteins may still be trapped in the vesicles destined for the golgi which would need to be sent back to the ER. The KDEL sequence would be recognized by the empty KDEL receptor which would bind to the adaptor proteins then to the COPI proteins which would help it bud off and bind to the ER
Retrograde- going backwards
63
Protein that direct vesicles to the right place for docking: Rabs & Tethers
The vesicle recognizes its target membrane with snares and rabs and tethers
Active Rab-GTpases are present on both vesicles and target membranes
If the right Rab binding/tethering protein is present on the target membrane the vesicle will tether- tethering protein will bind onto the rab
This brings the vesicle close enough proximity to begin membrane fusion to allow snares to interact
Snares bring vesicles even close to membrane causing it to fuse
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Proteins that mediate vesicle fusion: SNARES
v-SNAREs (on vesicle membrane)
t-SNARES (on target membranes)
Must have at least one SNARE on each membrane and 4 distinct SNARE coils to mediate fusion
Hydrophobic amino acids on surfaces on one side of alpha helix that zip together to coil together with another alpha helix snare
Molecular model showing SNARE interactions forcing lipid bilayer together
Outer leaflet starts to flow into the outer leaflet of PM
Protein orientation (N-C) does not change with vesicle fusion
Cytosolic side of membrane proteins remains on the cytosolic side
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Vesicle budding from the donor membrane:
| Key components involved in budding:
Membrane coat (Clathrin, COPI, COPII, and adaptor proteins)
GTPase enzyme that when bound to GTP helps to regulate assemble membrane coat
Proteins that “pinch off” the vesicle (ex. Dynamin for clathrin coats)
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Vesicle docking and fusion to the target membrane:
| KEY components involved in fusion:
Proteins that direct vesicles to the right place (Rab GTPases, tethering proteins)
Proteins that bind to target membrane and mediate fusion
SNARE proteins: v-SNAREs in vesicle (donor) membranes
t-SNAREs in target membranes
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Golgi: Major Themes
1. orientation/structure of the golgi
2. glycosylation/modification in the golgi
3. Transport through the golgi
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Golgi apparatus is a series of flattened stacks called cisternae
Cisterna (singular) or cisternae (plural): fluid-filled sac
| E.g. the cis, medial, and trans cisternae of the golgi apparatus
69
mammalian golgi vs. plant cell golgi
The mammalian golgi is a perinuclear ribbon/stack around nucleus
Plant cells have small individual golgi stacks moving all around the cell (“stacks on tracks”)
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cis=
trans=
cis to trans=
trans to cis=
cis= on the same side
trans= across, beyond, through
Cis to trans= anterograde
Trans to cis = retrograde
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Functions of the golgi
Recall that glycosylation is initiated in the ER by covalent attachment of the oligosaccharide tree (14 sugars)
Glycosylation continues in the golgi (removal, addition, and modification of sugars)
Additional processing for packaging and sorting occurs in the golgi (coding for this has to be in the protein itself)
Note: in plants and fungi, the golgi has an additional role: the synthesis and secretion of many of the polysaccharide components of the plant cell wall.
72
Early glycosylation of proteins in the ER
1. Oligosaccharides are added to dolichol (phospholipid) anchored in the cytosolic face
2. A flippase enzyme moves this structure into the ER
3. This oligosaccharide structure is transferred from dolichol to the polypeptide
The transferrase recognizes “Asn-X” sequences (where “X” is Ser or Thr) and links the oligosaccharide to Asns
Oligosaccharide added in ER
4. In ER, golgi oligosaccharide trimmed
5. In Golgi new sugars added to oligosaccharide one at a time
73
Different protein modifications take place in different compartments of the golgi:
The localization of each processing step was determined by combination of techniques:
Sub-fractionation of golgi apparatus- separating different cisternae of golgi and figuring out what the components are
EM after staining with antibodies specific for some processing enzymes to see where the proteins are located
74
Summary: glycosylation, packaging, & sorting
1. Oligosaccharide transferred to new protein and trimmed
2. Glycoprotein packaged into vesicle and sent to golgi
3. Based on protein structure, specific sugars are added by transferase enzymes
4. Based on its overall structure each glycoprotein can be recognized packaged into a vesicle and sent to a different compartment
75
Types of proteins in golgi:
| Cargo proteins:
Just passing through, moving on to other destinations on the endomembrane system
E.g. proteins to be secreted, sent to PM, sent to lysosome etc..
Proteins move through golgi stack in cis to trans direction, they appear..
First in the cis cisterna
Then in the medial cisternae
Then in the trans cisternae
Then into the trans golgi network (TGN) to be packaged into vesicles and sent to its next destination
76
Types of proteins in golgi:
| Resident proteins:
That have full time jobs in the golgi apparatus
E.g. glycosyl transferase, other protein-modifying enzymes to the proteins that are passing through
Different resident proteins are present in different cisternae of the golgi apparatus
Retention signal to keep protein in specific cisternae - labelled
77
There are 2 major transport models
Vesicle transport/vesicular transport model, and Cisternal maturation: model transport through golgi
Both models exist and are used by cells. The type of transport varies depending on the cargo and function of the cell
78
Vesicle transport/vesicular transport model:
Cargo is carried forward cis trans in vesicles
Cargo proteins are moved from one cisterna to the next (cis to trans) by transport vesicles
Cisterna remain mostly the same
Resident proteins that may get trapped in the vesicles must travel back in retrograde direction to get back to the correct cisternae
Resident proteins stay in place in cisternae (chained to the bench)
Assembly line like model- cargo proteins are moved from one work-station to the next
79
Cisternal maturation: model transport through golgi
Cargo is not carried in vesicles, instead the vesicles contain resident enzymes moving in reverse
Vesicles carrying cargo proteins fuse to form cis-cisterna which moves forward
Cargo proteins remain in the cisterna
When a new cis-cisterna forms, the old cis-cisterna becomes the medial-cisterna etc
Eventually trans-cisterna breaks up to form vesicles
Resident proteins must move up the stack (trans to cis) as cisternae mature and move down the stack (cis to trans) to maintain a relative position within the golgi stack
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Chaperone proteins (normally active in ER to help fold proteins) would be expected to contain____signal sequence
Resident proteins such as chaperones need to be returned to the ER. this happens through:_____
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KDEL, retrograde and COPI coated vesicles
81
Predict what is being transported in the vesicles moving between the cis and medial cisternae
Mature resident enzymes that function in the cis cisternae- retrograde
Cargo proteins moving from cis to medial cisternae- anterograde
82
Proteins in the nucleolus are not glycosylated
These proteins are brought directly into the nucleolus from the cytosol
Glycosylation happens in the ER
83
Order of steps of secretion of glycoproteins
1. Ribosome binds to mRNA in cytoplasm
2. Co-translational insertion of proteins into ER
3. Enzyme transfers oligosaccharide ‘core’ from dolichol to protein
4. Oligosaccharide has sugars trimmed and added by glycosyl
5. Protein is packaged in trans-golgi into secretory vesicles
84
The post -golgi region of the endomembrane system includes:
Traffic from the golgi
Exocytosis (i.e. secretion) = movement of molecules to the plasma membrane or out of the c4ell
Targeting of newly synthesized resident of endocytic/lysosomal pathways
Traffic from the plasma membrane
Most commonly destined for degradation in the lysosome
May also include some recycling of receptors, etc. back to the plasma membrane
85
3 main pathways for materials that leave the golgi going in anterograde motion toward the cell surface
1. Signal mediate diversion to lysosome
2. Signal-mediated diversion to secretory vesicles (for regulated secretion)- held and released upon signal from the trans golgi network (exocytosis)
3. Constitutive secretory pathway- default for any other proteins in the endomembrane system that have no other signal (exocytosis)
86
Change in pH as you move through endomembrane system
ER: neutral side of pH along with cytosol
As we travel through golgi, the cisternae become more and more acidic
Lysosomes: very acidic
Change in acidity is important for how the proteins are sorted into locations in the endomembrane system
87
Two different ways cargo can be exocytosed
1. Constitutive secretory pathway
| 2. Regulated secretion
88
Constitutive secretory pathway
Happens all the time. No signal required for vesicle release
Vesicle formation generally does not require coat proteins also called the default secretory pathway
Clathrin is NOT needed though it is generally need for golgi to PM vesicles
Materials that we wanted transported often, so purpose:
To supply PM with new lipids and proteins
Expansion of PM prior to cell division
Refresh old lipids
Contribute to extracellular matrix
Secrete nutrients and/or signal for other cells (e.g. hormones needed for cell survival)
89
Regulated secretion
Vesicles only released when a specific signal is received (binding of hormone, action potential, etc.)
pH is thought to play a role in the formation of vesicles as these proteins tend to play a role in the formation of vesicles as these proteins tend to aggregate at low pH
Soluble in a neutral pH and they start clumping together in lower pH
Changes in folding as certain parts will not like to interact with positive environment
Protein clumps are ready for vesicle transport
Purpose: have proteins rapid and ready for release
90
Experimental methods used to gather evidence on the Secretory pathway
1. Autoradiography: not widely used anymore; uses radioactively labeled macromolecules to locate specific compounds in the cell; uses film
2. Biochemistry
3. Studying genetic mutants with mutations in key secretory proteins
E.g. yeast (saccharomyces cerevisiae) secretory (sec) mutants
4. Secretory proteins genetically fused to green fluorescence protein (GFP): labeled proteins can be tracked in live cells using confocal microscopy; proteins disperse when vesicle comes in contact with PM
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Experimental methods: using secretory (sec) mutants
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The secretory (sec) mutants were important in dissecting the proteins involved in secretion
Temperature-sensitive mutants are only blocked at higher temperatures
At lower temperatures the protein functions normally
Way to maintain mutants without killing the cell and study its effects
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Fusion of vesicles with their target organelle is dysfunctional
Build up of vesicles within the cell
Find out the dysfunction; determine what mutation
Snares could have a mutation; fusion of vesicles are impaired
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mutation: endomembrane protein build up in the cytosol
All of the proteins in the endomembrane system is in the cytosol
A mutation in the SRP (binds to signal sequence found in all proteins targeting the endomembrane system) so that the protein cannot transport into the ER lumen
Defective function: transport into the ER
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mutation: endomembrane protein build up in the RER
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Receptors can’t bind to cargo in ER at higher temperature
For class B, the build up could be because a lack of COPII (which builds coats for transfer to golgi) so that it can’t properly form vesicles to send to golgi
Defective function: budding of vesicles from the RER
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mutation: endomembrane protein build up in the transport vesicles between ER and golgi
Problem with v or t-snares, or protein on the outside of the vesicle that allows for it to dock. If there is a mutation here, the vesicle will not be able to get its contents to the golgi
Defective docking and fusion (SNARES, Rabs, tethers) for targeting and fusion of transport with golgi to target membrane
96
More likely to have a mutation in the machinery involved in the mechanism that gives the observed phenotype than a mutation in every gene.
Unlikely that all secreted proteins in a cell have the same mutation, that causes a change in conformation/sequence. More common for a defect in protein machinery involved in ER entry (SRPs, etc.), vesicle formation and budding (coat proteins, scission, proteins, adaptor proteins, etc.)
97
Post Golgi transport: the lysosomal pathway
| Targeting to the lysosomes requires
1. Signal (mannose-6-phosphate)
| 2. Receptor (mannose-6-phosphate receptor)
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Post Golgi transport: the lysosomal pathway
Targeting to the lysosomes requires:
Signal (mannose-6-phosphate)
Mannose-6-phosphate (M6P) is the targeting signal for lysosomal proteins
Phosphorylated sugar on 6th carbon
Modification found on lysosomal proteins
Amino acid sequence determines whether they are phosphorylated
First glycosylation step in ER then more modification steps in the golgi
Phosphorylation of mannose happens in the golgi by one of the golgi resident proteins
Carbohydrates (including mannose) are added to proteins in ER and Golgi
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Post Golgi transport: the lysosomal pathway
Targeting to the lysosomes requires:
Receptor (mannose-6-phosphate receptor)
Cargo destined for lysosomes are recognized by M6P receptors and packaged in clathrin-coated vesicles
Adaptin proteins that recruit the clathrin coat
Freshly budded vesicles from the TGN, containing synthesized acid hydrolases, fuse with late endosomes to form mature lysosomes. This is the site of active digestion
Enzymes get released into the late endosomes
Lysosomal proteins are released from receptors with the late endosome and the receptors are recycles
pH allows lysosomal proteins to dissociate from from their cargo receptors; protein changes conformation in lower acidity and are now able to dissociate from cargo receptors
Receptors are recycled back to the trans golgi network
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Further processing allow lysosomal enzymes (e.g. Cathepsin D) to become active only in mature lysosomes
1. Removal of pro-peptide, which keeps the protein from folding to its final conformation
2. Removal of phosphate group
3. Lowering of pH
4. Final processing of peptide into subunits
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Lysosomes:
Digestive enzymes to break down macromolecules into monomers:
E.g. nucleases, proteases, glycosidases, lipases, phosphatases, sulfatases, and phospholipases
H+ pump to maintain acidic environment
Lysosome in the liver cell of a rat; vacuoles in plant cells also function as lysosomes
All resident (normally get tagged there) lysosomal proteins are glycoproteins
All resident lysosomal proteins have to have gone through the ER which are glycosylated
The lysosomes do not digest themselves due to heavily glycosylated proteins that act as a lining for the membrane
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Lysosomal Diseases
There are as many as 40 human diseases caused by defects in the lysosomal pathway
Example include:
Storage diseases:
Niemann-Pick diseases (lipids cannot be degraded)
Tay-Sachs diseases (neuronal lipids cannot be degraded)
(frame-shift mutations in lysosomal enzymes results in improper splicing of the mRNA)- die in early childhood
Hunter Syndrome and Hurler’s disease (oligosaccharides cannot be degraded)
Autoimmune diseases
I-cell diseases
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Inclusion-cell (I-cell) disease
In patients with I-cell disease, cells do not digest material in their lysosomes and undigested material accumulates as “inclusions”
Fibroblasts do not digest materials in their lysosomes
Lysosomal enzymes are found in the patients blood
A single gene defect is found in the enzyme which adds phosphate to mannose-6-phosphate oligosaccharides in golgi
Primary defect: Mannose-6-phosphate receptor in the TGN doesn’t recognize enzymes for sorting due to lack of Mannose-6-Phosphate
Lysosomal enzymes are found in the blood because lysosomal enzymes are secreted as secretion is the default pathway
104
Summary: Lysosomal Targeting
Targeting to the lysosome begins in the rough ER (rER) during/after cotranslational insertion
In the rER, these proteins have a core sugar transferred to them from dolichol. This is trimmed and processed so that it consists primarily of mannose sugars
In cis-Golgi, an enzyme transfers a phosphate to the mannose sugars so that they become mannose-6-P. This is the tag- the lysosomal marker. Only lysosome-destined proteins carry the M6P tag
In the TGN the receptors for M6P bind to all the proteins containing M6P and gather them together into a vesicle- with clathrin coat
Lysosome-destined vesicles fuse with late endosomes with a lower pH environment- where the cargo dissociates from the receptor and the receptor is recycled (sent back to the TGN)
Phosphate is removed from the lysosomal protein by another enzyme (phosphatase) before the protein can become functional in the lysosome
105
Macrophage secretes the equivalent of 100% of its plasma membrane each half hour via secretion. __% of its membrane is being brought back via endocytosis.
100, The surface area of the plasma membrane is maintained through a balance of secretion (new membrane) and endocytosis (take-up of membrane)
106
Three types of endocytosis:
1. Phagocytosis - specific/selective
2. Pinocytosis = constitutive (on all the time) endocytosis; a continuous process where cells remove excess membrane added by exocytosis allowing recycling of the PM
3. Receptor mediated - specific/selective
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Phagocytosis
specific/selective
In mammals: certain types of white blood cells act as “professional phagocytes”- cells of immune system
1. Macrophages
2. Neutrophils
3. Dendritic cells
Ingest cellular debris (aging and dead cells) and invading microbes
Ingesting an invading bacteria
Role in:
Defense - fight off any sort of pathogens and gets digested
scavenging/cleaning up
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Pinocytosis
constitutive (on all the time) endocytosis; a continuous process where cells remove excess membrane added by exocytosis allowing recycling of the PM
Non-specific
This process is nonselective
Fluid and macromolecules from the extracellular region are taken up without regard to the type or concentration of molecules present (e.g. salts)
There are no specific receptors for uptake of macromolecules
May or may not use coated vesicles
Exocytosis & endocytosis balance each other
Advantages: maintain cell size, constant, possible faster process
Disadvantage: cannot control what goes in whether it is bad or good
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Receptor mediated endocytosis
specific/selective
Receptors used to collect specific extracellular compounds
Receptors target specific compounds
Clathrin used for vesicle formation (bends membrane)
After vesicles form, coat proteins dissociate from the vesicle
Example: low-density lipoprotein (LDL)
LDL is a complex that makes lipids/cholesterol water soluble so they can be carried in the bloodstream
Receptors used to bring LDL into cells
110
Receptor mediated endocytosis: LDL as an example ligand
Adaptor proteins that bind to the tails of the tails and the adaptor proteins recruit the clathrin coat
Vesicle forms and coat is removed and fuses with the endosome
At a lower pH, the ligand is released due to the changes in protein conformation; the cargo is released, and the receptor is recycled, budded off the LDL receptors return to plasma membrane to pick up more cargo
Transport vesicles coming off the golgi with lysosomal enzymes will fuse with the endosome and that matures into the lysosome; lysosome with lysosomal enzymes and cargo molecules and those components get digested into monomers and the monomers get exported back into the cytosol for use
In the case of LDL; the monomers are free cholesterol that get exported into the cytosol for use
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In endocytosis where would M6P-containing glycoproteins enter this pathway?
Trans golgi network to the late endosome to mature into a lysosome
Receptor mediated endocytosis & lysosomal pathway from TGN
Recycling of cell surface receptors and M6P receptors by endosomes
Green circles= cargo molecules from extracellular region
Red squares = lysosomal enzymes (carried by M6P receptors from trans Golgi to late endosomes
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Endosomes:
| Endosomal compartments act as a sorting station for....
1. proteins/material that enter the cell and are destined for:
Lysosome- for full digestion
Cell surface- receptors get sent back to respective locations
Other endomembrane compartment
2. Proteins that leave the TGN destined for the lysosomes
113
Predict how increasing acidity could affect the structure of the proteins as they move through the endomembrane system.
Increasingly acidic environment would modify protein structure especially if there are basic/acidic R-groups in the polypeptide chain; protein folding would be different.
114
Why might changes in acidity be important to endomembrane function
Acidity helps proteins to aggregate to certain organelles (e.g. lysosome) which helps with packaging of cargo to form vesicles for transport.
115
Mitochondria and chloroplast facilitate the flow of biological energy
Chloroplast creates carbon bonds from light energy
| The carbon bonds are oxidized and turned into ATP to be used throughout the cell
116
Mitochondria_____________ is highly folded increasing its surface area. Though chloroplasts contain an_____________, it is not highly folded
inner envelope membrane
117
_____________ have a porous outer membrane and allows molecules to diffuse across freely but not porous inner envelope membrane
Chloroplast and the mitochondria
118
__ cannot be transported across the envelope membrane out to the rest of the cell from the chloroplast. The ___ made by chloroplast stays in the chloroplast. It is used to ....
ATP; fix carbon dioxide to make sugars. The sugars are what leaves the chloroplast.
119
Chloroplast, mitochondria contain DNA and ribosomes
Chloroplast and mitochondria contain remnant genomes carried on circular chromosomes, which reflect the evolutionary origins of the organelle
120
No functional ribosomes in the ____ as all its proteins are translated in the cytoplasm
nuclei ; the subunits are put together in the nucleus but they are not functional
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Endosymbiont Theory
Several lines of evidence suggest that mitochondria and chloroplasts originated from other prokaryotic cells:
Similar size and morphology
Divide by fission- just like bacterial cells
Contain own DNA and ribosomes- from genome of prokaryotes
Eukaryotic cell engulfed by endocytosis prokaryotic cell (mitochondria, chloroplast)
Double membrane (2 lipid bilayers) ; outer membrane that got derived from the cell membrane do the host, and an inner membrane that still resembles membrane of prokaryote
Reduction in size of organelle genome by: gene loss, some of the mitochondria and chloroplast gene got transferred to nucleus
The majority of all mitochondrial & chloroplast proteins are encoded by nuclear genes and these proteins need to be imported into organelles; mitochondria and chloroplasts are semiautonomous
Semiautonomous - they can kind of remain in the cell independently but not completely
Their proteins are encoded by genes in itself (mitochondria, chloroplast) as well
Mitochondria and chloroplast cannot survive if isolated from the cell
Chloroplast proteins are synthesized in the stroma and cytoplasm- depends if they are nuclear-encoded or chloroplast-encoded
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Mitochondrial/chloroplast import also uses specific targeting sequences
Found at the N-terminus of the protein (~18aa)- N-terminal signal
Recognized after the protein has been released from the ribosome (fully translated) but chaperones inhibit it from folding completely allowing the signal sequence to be exposed and recognized
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Targeting of nuclear-encoded mitochondrial matrix proteins (TOM/TIM)
Protein containing a specific N-terminal mitochondrial signal sequence is recognized and bound by receptors; unfolded during entry and refolded on the organellar side of the membrane by chaperones
Signal binds to receptor in the outer membrane and brings the polypeptide to a TOM complex
Translocation through TOM complex; there is a corresponding TIM complex
TOM/TIM kind of form a pore for the polypeptide to be translocated through the double membrane and into the matric
If mitochondrial protein could be removed from the mitochondria after import, it would not be able to return to the mitochondria by import in the cytosol as the signal sequence is cleaved off with signal peptidase
124
Targeting of nuclear-encoded thylakoid lumen proteins (chloroplast) (TOC/TIC)
N-terminal chloroplast signal sequence recognized in the same manner
Chloroplast signal that is recognized by receptor complex and directs the translocation of the TOC/TIC complexes similar to
TOC- translocator outer chloroplast; TIC- translocator inner chloroplast
Chloroplast polypeptide ends up in the stroma
Signal sequence is removed
If the polypeptide is destined for the thylakoid, you also need another thylakoid signal sequence right after the chloroplast signal sequence
The thylakoid signal sequence is exposed upon cleavage of the chloroplast signal sequence; the thylakoid signal sequence will allow it to enter the thylakoid and once the protein enters the thylakoid, another signal peptidase cleaves off the signal sequence and full folding
Once in final destination, polypeptide undergoes full folding
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Mitochondria produce ATP for the cell
Mitochondria is the powerhouse of the cell
Mitochondria are dynamic in size & shape, and move around the cell
They often fuse and form elongated network through the cytoplasm
Responsible for most of the respiratory steps
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Oxidation of glucose
1. Glycolysis in cytosol (anaerobic-does not require oxygen)
Glucose is oxidized to a molecule called pyruvate and generate ATP from it
Also generate electron carriers called NADH (NAD is reduced to NADH)
Pyruvate, NADH, and electrons enter the mitochondria into the matrix
ATP generated by substrate level phosphorylation
Pyruvate (enters mitochondrion for citric acid cycle)
Electron donors (enters mitochondrion for electron transport chain)
2. krebs/citric acid/TCA Cycle
Pyruvate oxidized further to CO2 - controlled burn of pyruvate molecule
As you are breaking these bonds, we are releasing carbon dioxide
Transferring energy from broken bonds to the high energy bonds within ATP
Reducing electron carriers from NAD to FAD to NADH to FADH2
ATP equivalent produced (small amount)
Electron donors produced
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Oxidative phosphorylation
1. Electron donors drive electron transport chain (ETC) and produce proton gradient across the cristae membrane
Electron transport chain powers proton pumps in inner membrane of mitochondria
Causes inner membrane space to become quite acidic; create proton gradient
The protons then flow through these ATP synthases- mechanical pump which phosphorylate ADP to make ATP
Fatty acids also get metabolized by mitochondria
2. Proton gradient drives the production of ATP. requires oxygen as final electron acceptor to pick up protons and H20 is produced
128
____________is very acidic in the mitochondria
Intermembrane space
129
Chloroplasts contain chlorophyll: the primary light harvesting pigment found in plants
The ability to produce one’s own energy is a valuable skill
There are organisms that can steal chloroplasts from photosynthetic organism, algae
Egg masses of the spotted salamander Ambystoma maculatum, with green algae (oophila amlystomatis) in embryonic cells
Chloroplasts have double envelope: inner membrane and outer membrane
Thylakoid stacks- third membrane
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Chloroplasts are the most prominent members of the plastid family of organelles
Plastids are also sites of synthesis for purines & pyrimidines, most amino acids & fatty acid in plants (other macromolecules)
Gravity sensing amyloplast from root tip- plastids; starch granules fall to lowest point in the plastid and so the cell knows gravity and orients itself; also storage of starch
An etioplast from a dark grown leaf- when there is no exposure to light
Chromoplast- contains other pigments
Plastids can differentiate according to the role they perform
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Fixation of carbon (from CO2) into sugars by the calvin cycle
Rubisco is considered to be the most abundant protein on earth; hugely important plant enzyme in carbon fixation
Rubisco-mediated is inefficient; 9 ATP and 6NADH to fix 3 CO2 molecules (each sugar needs 6 CO2 molecules)
Lots of energy in carbohydrate molecules
Huge energy requiring pathways
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Chloroplast catalyze a major conversion of energy
| Light-dependent reactions:
sunlight powers electron transport and production of high energy molecules
Charging of electrons that move through the electron transport pathway
Energy is used to reduce an electron carrier; NADP to NADPH
Energy transduction reactions (aka light reactions)
Light drives electron transport and powers proton pumps
Electron donors also produced
Proton gradient drives ATP production
Proton gradient is within the thylakoid lumen; protons move from the thylakoids to the stroma to power those ATP synthases
Sunlight activates photo centres which causes water to turn into H+ (protons) and oxygen
Movement of electron moves through the electron chain and is used to reduce NADP to NADPH
It is most acidic in the thylakoid stacks
Protons flowing through ATP synthase into the stoma, the mechanical energy is harnessed to add phosphate to ADP to make ATP
Protons move with the proton gradient (high to low concentration) through the head of ATP synthase
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```
Chloroplast catalyze a major conversion of energy
Light independent (dark) reactions (Calvin cycle):
```
high energy molecules are used to fix CO2 into sugars
Does not directly require sunlight; instead uses products that were generated in light-dependent reactions
ATP and NADPH, electron donors provide the energy to fix CO2 into sugars (calvin cycle)
The sugars made by photosynthesis can be used to by respiration to make ATP
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Summary of photosynthesis:
Light powers the electron transport chain and leads to the production of ATP and NADPH
Splits water to generate those electrons to get protons for the proton gradient
ATP and NADPH is used in the calvin cycle to fix CO2 to make sugars
135
ATP made in the chloroplast STAYS in the chloroplast; instead....
Sugars are the primary export
ATP made in photosynthesis is used to make sugars, not by cell directly
Sugars are taken up by mitochondria; the sugars are oxidized in the citric acid cycle to produce ATP
136
The flow of biological energy
Light energy used to fix CO2 into energy-rich sugars-energy is stored in the bonds created between molecules
Macromolecules (sugars, proteins, fats) are oxidized to release energy bonds are broken to make ATP
Energy stored in chemical bonds are released through oxidation
137
Streptomycin
an antibiotic that binds to chloroplast’s ribosomes and inhibits translation
Chloroplast cannot replicate its DNA
138
ER import signal
```
target-ER
co-translational recognition
protein amino acid sequence
hydrophobic
cleaved only if N-terminal
```
139
M-6-P signal
```
target-lysosome
post translational recognition
sugar signal
phosphorylated property
not cleaved
```
140
Mitochondrion signal
```
target- mitochondrial matrix
post-translational recognition
protein amino acid sequence
hydrophobic properties
cleaved
```
141
chloroplast signal
```
target- chloroplast stroma
post-translational recognition
protein amino acid sequence
hydrophobic properties
cleaved
```
142
thylakoid (within chloroplast)
```
target- thylakoid lumen
post-translational recognition
protein amino acid sequence
hydrophobic properties
cleaved
```
143
NLS
```
target-nucleus
post-translational recognition
protein amino acid sequence
basic (+ charged)
not cleaved
```
144
no signal=
cytosol
145
ER (N-terminal only)=
secreted (outside the cell)
| Signal peptidase inside ER is going to cleave off the sequence and the protein will be soluble inside ER
146
ER(N-terminal and Internal transfer sequence- start/stop) =
Plasma membrane
147
ER (internal transfer only)=
Plasma membrane
148
ER (any) + KDEL (located on protein region in lumen)=
ER
149
KDEL alone =
cytosol *can’t recognize KDEL if not inserted into ER first*
| KDEL only gets recognized in the endomembrane system
150
ER (any) + M-6-P =
lysosome
151
ER (N-terminal) +NLS =
secreted *nothing can recognize NLS in the EMS*
152
ER(internal transfer) + NLS =
Plasma membrane *can’t recognize NLS in the EMS*
153
Mitochondrion signal =
mitochondrial matrix
154
chloroplast signal =
chloroplast stroma
155
Chloroplast + thylakoid signal sequence=
thylakoid lumen
156
Protein Processing
```
Folding (all proteins)
Joining of subunits (form Quaternary structure)
Disulfide bonds (endomembrane only)
Proteolytic cleavage (endomembrane only)
E.g. Pro-insulin is cleaved into regular insulin in the secretory regulatory vesicles
Glycosylation (endomembrane only)
Glycosylation (endomembrane only)
Oligosaccharide tree is added to Asn residues when the “Asn-X’ sequence is recognized (X= Thr, Ser)
This tree may be cut down into smaller sugars in the golgi apparatus
```
157
Dolichol
the molecule that is holding onto oligosaccharide in the ER membrane
Protein transferase is going to transfer the tree onto Asn residue
158
how is the mannose sugar tagged?
Whole oligosaccharide tree on lysosomal protein in the ER that will get moved over to the golgi apparatus. Then, extra sugars that aren’t necessary will be trimmed, and then will phosphorylate the mannose
159
of internal transfers=
of internal domains
160
N-terminal orientation of polypeptide when internal ER signals only=
N-terminus has cytosolic orientation
161
N-terminal orientation of polypeptide when N-terminal ER signal ==
N-terminus has non-cytosolic orientation
162
C-terminal orientation when last transfer sequence is STOP
C-terminal cytosolic
163
C-terminal orientation when last transfer sequence is START
C-terminal non-cytosolic
164
Vesicle Transport: COPII:
ER to cis golgi
| Need adaptor protein + GTPase
165
Vesicle Transport: COPI:
```
Retrograde transport: Cis Golgi to ER
Transport between golgi cisternae
Secretory pathway: trans golgi network to PM for constitutive secretion in some cases
adaptor protein included in coat
need GTPase
```
166
Vesicle Transport: Constitutive secretion
mostly does not need coat protein but sometimes COPI
167
Vesicle Transport: Regulatory secretory vesicles
no coat protein; protein aggregate in the trans golgi network
168
Vesicle Transport: Clathrin coat:
Receptor-mediated endocytosis, lysosomal proteins
Lysosomal pathway: golgi to lysosome via late endosome
Endocytic pathway: plasma membrane to early endosome
Need adaptin + Dynamin + GTPase
169
Cisternal Maturation Model
Cisternae are constantly being formed from and broken down into vesicles
Golgi resident proteins move in retrograde fashion via vesicles
As the cisternae move, it will mature and decrease in pH
Cisternae containing processed proteins move in anterograde fashion to effect to correct cisternae
Cis-golgi network becomes cis cisterna, then medial cisterna, then trans cisterna, then TGN
Cargo proteins stay in the same cisternae and moves with it
170
Vesicular transport model
Cisternae stay constant in the same position
Golgi resident proteins remain in the exact same cisternae
Processed proteins move in anterograde fashion via vesicles
171
Endomembrane Pathways
| Secretory:
Constitutive: occurs in all cells, releases any endomembrane proteins without other signals
Regulated: occurs in specialized cells, requires a signal transduction to trigger release of contents (ex. Nerve cells receiving action potential to trigger release of neurotransmitter)
172
Endomembrane Pathways
| Lysosomal:
Newly synthesized proteins with M-6-P sent from TGN to lysosome via endosomes using clathrin coated vesicles
173
Endomembrane Pathways
| Endocytic:
External components taken up and sent from plasma membrane to lysosome via endosomes
Phagocytosis (specialized cells- eating up bigger things), pinocytosis (all cells- opposite direction but kind of like constitutive), receptor-mediated endocytosis (clathrin coat, going to lysosome)
174
Endomembrane Pathways
| retrieval/recycling:
Works in opposition of other pathways to maintain balance in size of membranes and location of proteins
Retrograde movement to recover some other parts of endomembrane system
175
Early endosome to late endosome maturation:
pH decrease; proton pumps that pump in protons in to the endosome
Aggregation of existing proteins
176
Receptor-mediated endocytosis + lysosomal pathway
| +Early endosome to late endosome maturation:
Early endosome forming from parts of previous endosomes and can include vesicles from the plasma membrane with vesicles coming from TGN potentially with proton pumps
pH decrease; proton pumps that pump in protons in to the endosome
Lower pH allows cargo from PM to dissociate from its receptors
Receptors will be recycled back to PM
Aggregation of existing proteins
Late endosome will fuse with existing lysosome to form new lysosome
177
M-6-P receptor:
only used to bind to proteins with M-6-P signal and that will target the lysosome by bringing it to the late endosome.
M-6-P receptor provides the proper signalling for the clathrin coat to form and for the vesicle to be targeted to the right place
178
Lollipop head of ATP synthase point into the _________ and the____________.
These areas have a higher pH.
___________ and __________ will have lower pH due to protons being pumped in them.
mitochondrial matrix, stroma of the chloroplast, Thylakoid lumen, intermembrane space of mitochondria
179
Protons flow back across membrane through ATP synthase to generate ATP in _________
Matrix or Stroma
