BIS104 MT2

Dinesh-Kumar Midterm 2 UC Davis

Lecture 9 04/21

Triton X-100

• Solubilizes membranes while preserving protein structure

• Replaces lipid bilayer with detergent micelles

• Allows investigation of protein topology

SDS Detergent

• Stronger than triton X-100

• Harsh, denatures proteins completely

Trypsin Digestion Experiment

• Purpose: Determine orientation of membrane proteins

• Experimental Setup:

  • Homogenize cells/ER → microsomes

  • Add Triton X-100

  • Treat w/trypsin

  • Analyze SDS-PAGE

Trypsin Digestion Experiment Outcomes

• Protein fragments indicate which portions were exposed

• size change correlates with amount of protein digested

  • Large size reduction → large extracellular portion

  • little or no size change → mostly intracellular

• Special cases

  • Peripheral proteins inside membrane: No size change

  • Peripheral proteins outside membrane: Completely digested

Selective permeability

Allows for separation and exchange of materials across the plasma membrane

Types of Diffusion Across Membranes

1. Simple Diffusion: Small molecules (O₂)

2. Passive Transport via Channels

   • Voltage-gated Na+ channels/leak channels

   • Voltage-gated K+ channels/leak channels

   • Rapid ion flow

3. Facilitated Diffusion via Transporters

   • Glucose (GLUT1, GLUT4)

   • Requires binding and conformational change

4. Active Transport via Pumps

   • Na+/K+ ATPase pump

   • Moves substances against concentration gradients

   • Requires ATP hydrolysis

Voltage-gated channels

conformational state depends on difference in ionic charge on 2 sides of the membrane

ligand-gated channels

conformational state depends on binding of specific molecule (ligand)

Mechanism-gated channels

conformational state depends on mechanical forces that are applied to the membrane

Potassium Ion Channel Specificity

• Composed of 4 subunits

• Pore region (P-domain) containing GYGVT peptide

• Pore size matches K+ ions precisely

• Sodium ions too small to interact effectively

iClicker

You run proteins isolated from untreated control cells to trypsin treated cells on SDS-PAGE and transfer them to a membrane. You can detect 5 different proteins for which you have specific antibodies. What type of membrane protein is protein #5?

A. Transmembrane protein with equal domains both inside and outside the cell

B. Transmembrane protein with a large domain on the outside of the cell

C. Peripheral cytoplasmic protein

D. Peripheral extracellular protein

E. Transmembrane protein with large domain inside the cell

C. Peripheral cytoplasmic protein

Reasoning: No change after trypsin treatment, meaning no cleave and no access to inside of cell where cytoplasm would be.

iClicker

You run proteins isolated from untreated control cells to trypsin treated cells on SDS-PAGE and transfer them to a membrane. You can detect 5 different proteins for which you have specific antibodies. What type of membrane protein is protein #1?

A. Transmembrane protein with equal domains both inside and outside the cell

B. Transmembrane protein with a large domain on the outside of the cell

C. Transmembrane protein with large domain inside the cell

D. Peripheral cytoplasmic protein

E. Peripheral extracellular protein

B. Transmembrane protein with a large domain on the outside of the cell

Reasoning: Protein becomes very small, making a lot of protein accessible to trypsin, making it outside of the cell

iClicker

You run proteins isolated from untreated control cells to trypsin treated cells on SDS-PAGE and transfer them to a membrane. You can detect 5 different proteins for which you have specific antibodies. What type of membrane protein is protein #3?

A. Transmembrane protein with a large domain on the outside

B. Transmembrane protein with large domain inside the cell

C. Transmembrane protein with domains both inside and outside the cell

D. Peripheral cytoplasmic protein

E. Peripheral extracellular protein

B. Transmembrane protein w/large domain inside the cell

Reasoning: Protein moved towards bottom when treated with trypsin

Lecture 10 04/23

Ion channels

• passive transport, no energy required

• rapid movement (millions of ions/sec)

Facilitated Diffusion by Transporters

• slower than channels (hundreds to thousand ions/sec)

• Requires selective binding and conformational change

Facilitated Diffusion: Glucose Transporter

• Glucose binds, transporter changes shape to release glucose inside cell

• Insulin regulates GLUT4 (increase in blood glucose levels triggers secretion of insulin, stimulating the uptake of glucose)

• Insulin triggers transporter to move to cell surface (membrane)

Active Transport

• Maintains an imbalance of ions across the plasma membrane (moves molecules against conc. gradients)

• Generate gradients

• Requires coupled energy (ATP hydrolysis, absorbance of light, electron transport, flow of other substances down their gradients)

Concentration Gradients of Na+ and K+

Na+: high outside, low inside

K+: low outside, high inside

Primary Active Transport: Na+/K+ ATPase Pump

• Requires K+ outside, Na+ inside and is inhibited by ouabain

• 3 Na+ out/ 2 K+ in per ATP hydrolyzed

• Involves conformational changes, dephosphorylation and phosphorylation (ion affinity) bc it’s a P-type pump

H+/K+ ATPase

• maintains acidic pH in the stomach for digestion

• Histamine signaling moves pumps to membrane

• Treatments for acid reflux:

  • Antacids (Tums)

  • Histamine receptor blockers (Pepcid, Zantac)

  • Proton pump inhibitors (Prilosec)

Acid Production in stomach

Channels, Pumps, and Transported all work together to help with CO2 uptake, ion exchange, K+ cycling)

Protein Sorting Pathways: Co-translational

• part of protein is synthesized

• taken to ER to complete synthesis

• Sorted to ER, Golgi, lysosomes, plasma membrane, or secreted into extracellular space

Protein Sorting Pathways: Post-translational

Fully synthesized proteins sorted based on targeting signals

Nuclear Localization Signals

• Identified using SV40 virus large T-antigen studies

• Specific sequence (rich in lysine & arginine) directs nuclear entry

• Presence of basic amino acid-rich signal suggests nuclear localization

• Experimental approaches include:

  • deletion and point mutation experiments

  • fusion to GFP to test sufficiency

Lecture 11 04/28

SV40 large T antigen experiment

• nucleus alone is not sufficient for nuclear import and T antigen cannot enter (when it has no lysate)

• Ran’s GTP/NDP state cycling is key for transport through the nuclear pore complex for large molecules

• Ran is a GTPase function

  • Ran-GTP (active)

  • Ran-GDP (inactive)

• Ran-GAP helps hydrolyze GTP → GDP

• Ran-GEF helps Ran exchange GDP → GTP

Nuclear Import Process

1. Cargo Recognition in Cytoplasm

   • Protein A (w/NLS) binds to Importin-α

   • Importin-α then recruits Importin-β → forms a trimeric complex (Protein A + α + β)

2. Transport Through Nuclear Pore

   • whole complex moves through the nuclear pore complex into the nucleus

3. Dissociation in Nucleus

   • Inside the nucleus, Ran-GTP binds to Importin-β, causing it to release

   • Then, CAS binds to Importin-α in the presence of Ran-GTP, helping release the cargo (Protein A) and exporting α

4. Recycling to Cytoplasm

   • α-CAS-Ran-GTP and β–Ran-GTP complexes diffuse back to the cytoplasm

5. Reset by Ran-GAP

   • In the cytoplasm, Ran-GAP converts Ran-GTP → Ran-GDP, which releases Importins

   • Ran-GDP goes back into the nucleus where RCC1 (Ran-GEF) regenerates Ran-GTP

Nuclear Export Process

1. Cargo Binding (in the nucleus)

   • Exportin binds to the cargo protein with an NES

   • At the same time, Ran-GTP binds to exportin

   • Forms trimeric complex (Cargo + Export + Ran-GTP)

2. Translocation

   • Complex moves through the nuclear pore complex into the cytoplasm

3. Cargo Release

   • Ran-GAP in the cytoplasm converts Ran-GTP → Ran-GDP

   • This causes exporting to release the cargo protein

   • Exportin and Ran-GDP separate

4. Recycling

   • Exportin and Ran-GDP return to the nucleus

   • In the nucleus, RCC1 (Ran-GEF) converts Ran-GDP → Ran-GTP, resetting the system

Control of nuclear import during T cell activation

1. Resting T Cell (low calcium)

   • In a resting T-cell, the conc. of calcium ions (Ca2+) in the cytosol is low

   • NF-AT is phosphorylated and resides in cytosol

   • ATP & active protein kinase maintain NF-AT in phosphorylated state, preventing entry into nucleus

2. Activated T cell (High Calcium)

   • When T cell is activated, Ca2+ conc. in cytosol increases

   • High Ca2+ levels activate calcineurin

   • Calcineurin dephosphorylates NF-AT, removing P groups

3. Nuclear Import

   • Dephosphorylated NF-AT exposes nuclear import signal

   • signal allows NF-AT enter the nucleus through nuclear pores

4. Gene Transcription

   • Once inside nucleus, NF-AT activates gene transcription

   • Leads to production of proteins necessary for T cell’s function in immune response

5. Nuclear Export and return to resting state

   • After T cell carried out function, Ca2+ levels decrease

   • NF-AT is re-phosphorylated by ATP and active protein kinase

   • Re-phosphorylation exposes nuclear export signal, causing NF-AT to exit nucleus and return to cytosol, ready for activation cycle

Signal Sequence Functions

lysine, arginine, proline, etc

iClicker

You are working with a protein, SUN1, which is typically

localized to the cytoplasm. If you add the PRRKKKRV

sequence to the beginning of the SUN1 protein, where would

you expect to find SUN1?

A. Cytoplasm

B. Mitochondria

C. Nucleus

D. ER

E. None of the above

C. Nucleus

Reasoning: Sequence is rich in arginine and lysine

iClicker

You are working with a protein, SUN1, which is typically localized to the cytoplasm. If you add the PRRKKKRV sequence to the middle of the SUN1 protein, where would you expect to find SUN1?

A. Cytoplasm

B. Mitochondria

C. ER

D. Nucleus

E. None of the above

D. Nucleus

Reasoning: post-translation, already made once put in

iClicker

In your research project, you have identified a protein that contains the 110VRRKKKRKP118 sequence. The fusion of this protein to YFP and observation using fluorescence microscopy indicate that this protein localizes to the nucleus.

Then, you mutated lysine residues at positions 113 to 115 to alanine, and the fusion of this mutant protein to RFP abolished nuclear localization.

What would you conclude from this experiment?

A. The sequence is sufficient to drive this protein into the nucleus

B. The sequence is necessary to drive this protein into the nucleus

C. Sequence is necessary and sufficient to drive this protein into the nucleus

D. None of the above

B. The sequence is necessary to drive this protein into the nucleus

Lecture 12 4/30

TOM Complex

Protein-import complex in the outer mitochondrial membrane that include receptor and channel (translocon of the outer membrane)

TIM Complex

Protein-import complex in the inner mitochondrial membrane (translocon of inner membrane)

Mitochondria Import Assay

STEPS:

1. synthesize messenger RNA in vitro

2. Make protein in test tube using translation mic (ribosomes, buffers, other factors)

3. Radioactively label the protein with S35-methionine

4. Incubate labeled protein w/isolated mitochondria

5. Energize mitochondria to facilitate protein uptake

6. Wash mitochondria to remove unbound protein

7. treat w/trypsin to digest proteins outside mitochondria

8. separate proteins using SDS-PAGE

RESULTS: Smaller protein size in mitochondria due to cleavage of N-terminal terminal targeting sequence

N-terminal targeting sequence summary

• The N-terminal targeting sequence is exposed early during protein synthesis.

• Cytoplasmic chaperones bind to the protein to prevent complete folding and keep the N-terminal signal accessible.  

• The TOM complex recognizes this exposed N-terminal sequence.

• The protein is unfolded as it is translocated across the mitochondrial membranes.

iClicker

You identified a new protein that contains a mitochondrial matrix targeting signal sequence. Where would you expect to find GFP if you fuse GFP to the N-terminus of the mitochondrial matrix signal sequence of the newly identified protein?

A. Mitochondria

B. Nucleus

C. Cytoplasm

D. ER

C. Cytoplasm

Reasoning: By placing GFP at the N-terminus, you're blocking the targeting signal from being first in line, which prevents recognition and import into mitochondria. As a result, the fusion protein fails to be imported into mitochondria and instead stays in the cytoplasm. Should be [Mitochondrial signal]-[GFP], not [GFP]-Mitochondria signal]

iClicker

You identified a new protein that contains a mitochondrial matrix targeting signal sequence. Where would you expect to find GFP if you put the mitochondrial matrix signal sequence of the newly identified protein in the middle of GFP?

A. Mitochondria

B. Nucleus

C. Cytoplasm

D. ER

C. Cytoplasm

Reasoning: Mitochondrial targeting signal sequence was placed in the middle of GFP. Needs to be placed at the N-terminus to be recognized, so protein will remain in the cytoplasm

iClicker

You identified a new protein that contains a mitochondrial matrix targeting signal sequence. Where would you expect to find GFP if you fuse GFP to the C-terminus of the mitochondrial matrix signal sequence of the newly identified protein?

A. Mitochondria

B. Nucleus

C. Cytoplasm

D. ER

A. Mitochondria

Reasoning: Targeting signal had to be correctly at the N-terminus to have been imported to the C-terminus of the mitochondrial matrix

TIM23

• located in inner membrane

• translocated proteins with N-terminal matrix-targeting signals

TIM22

• located in inner membrane

• inserts multipass inner membrane proteins

TOM

• located in outer membrane

• first entry point for all nuclear-encoded mitochondrial proteins

TIM9

• located in intermembrane

• chaperones hydrophobic proteins across the IMS to TIM22

TRP1

often used as a marker for gene disruption and plasmid construction bc mutants require tryptophan to grow (trp1 gene encodes synthesis of tryptophan)

iClicker

The yeast TRP1 gene encodes an enzyme that catalyzes the first step of tryptophan synthesis. TRP1 is a cytoplasmic protein. You are conducting an experiment in which you fuse the mitochondrial matrix signal sequence to the N-terminus of TRP1 (mitoMatrix-TRP1). Cells carrying mitoMatrix-TRP1 were grown in the absence of tryptophan. Most cells died, but some grew. Why did some cells grow in the absence of tryptophan?

A. Because of a mutation in the TRP1 gene

B. Because of a mutation in the Oxa1 gene

C. Because of a mutation in the Tim22 gene

D. Because of a mutation in the Tim23 gene

E. Because of a mutation in the Tim9 gene

D. Because of a mutation in the Tim23 gene

Reasoning: TIM23 disrupts import of mitoMatrix-TRP1 fusion into the mitochondrial matrix, so fusion protein stays in the cytoplasm, where TRP1 can function normally.

Lecture 13 5/2

Endomembrane System

• Formed by ER, Golgi complex, endoscopes, lysosomes, vacuoles that act as a coordinated unit

• Distinct compartments bound by membrane barriers and contain specialized proteins for particular activities

Autoradiography

• visualizes biochemical processes by radioactively labeling molecules

• Showed ER as site where secretory protein synthesis occurred

• Palade and Jamieson incorporated radio labeled amino acids into pancreatic enzymes and were able to localize the cellular proteins

George Palade’s “Pulse-Chase” experiment

1. Pulse: Pancreatic cells were exposed to radioactive amino acid (S35-met) for a short time to label newly synthesized proteins

2. Chase: Cells were washed and incubated with non-radioactive amino acids to stop any new proteins from being labeled

3. Fixation and EM Imaging: At different time points during chased, cells were examined using electron microscopy and autoradiography

Results demonstrated secretory pathway of:

• Rough ER → Golgi → Vesicles → Plasma Membrane (secretion)

Using Fluorescent Proteins in Endomembranes

• GFP tagging allows microscope viewing of protein movement in living cells

• Production and movement of viral proteins can be monitored

5 Clones of Sec Mutations

• Sec A mutants: cytoplasm → ER

• Sec B mutants: ER → vesicles

  • ER larger

• Sec C mutants: Golgi → vesicles

  • excess vesicles from ER

• Sec D mutants: vesicles → plasma membrane

  • protein gets stuck in Golgi

• Sec E mutants: Plasma Membrane → secreted

  • protein gets stuck in vesicles btwn Golgi of pm

Rough ER (RER)

• ribosomes bound to its cytosolic surface

• flattened sacs (cisternae) connected to neighbors by helicoidal membranes

• is continuous with the outer membrane of the nuclear envelope

• starting point of biosynthetic pathway for secretory proteins

• 1/3 of proteins synthesized & released into ER lumen (co-translational translocation)

Smooth ER (SER)

• lacks ribosomes

• membranes are highly curved and tubular

• is continuous w/the RER

• calcium ion sequestration and regulated release

Gunter Blobel: Signal Hypothesis

• Translation of secretory proteins w/o ER = proteins are longer and contain signal peptides

• Translation w/ER present = shorter, processed proteins, confirming signal sequence was removed in the ER

Sec4

• Function

  • Rab GTPase localized on secretory vesicles

  • Helps direct vesicles to pm, interacts w/effector proteins to promote tethering, facilitates vesicle docking & fusion w/pm in GTP-bound active form

  • After fusion, hydrolyzes GTP to GDP & recycled

• Mutant

  • Vesicles cannot be properly targeted or docked to pm

  • post-Golgi secretory vesicles accumulate in cytoplasm

  • secretion blocked at final step after vesicle formation & Golgi exit

Sar1

• Function

  • small GTPase that plays critical role in COPII vesicle formation, mediating ER to Golgi transport in secretory pathway

  • Activated by Sec12

  • Recruits sec23/24

• Mutants of GDP-locked Sar1:

  • COPII vesicles cannot form

  • Proteins accumulate in ER, ER becomes enlarged, secretory pathway blocked

• Mutants of GTP-locked Sar1

  • COPII coats do not disassemble

  • vesicles bud but don’t uncoat (fusion w/golgi blocked)

  • vesicles accumulate

Sec12

• Function

  • Acts as GEF for Sar1 GTPase.

  • Catalyzes exchange of GDP for GTP on Sar1

  • GTP-bound Sar1 inserts into ER membrane and recruits COPII coat proteins (vesicle formation)

• Mutant:

  • Sar1 remains GDP-bound and inactive.

  • COPII vesicle formation is blocked

  • Enlarged ER due to buildup of proteins

Sec17

• Function

  • Disassembles SNARE complexes so they can be recycled

  • Works w/Sec18

  • Works after vesicle fusion

• Mutant

  • SNARE complexes remain locked after fusion

  • Future vesicle fusion events are blocked (SNARE proteins not recycled)

  • Block in vesicle trafficking throughout secretory pathway

iClicker

A sec12 mutant yeast cell lack vesicles and has a larger ER as compared to wild-type. A sec17 mutant has an excess of vesicles and a small ER as compared to wild-type. What would you predict a sec12, sec17 double mutant would look like?

A. Normal number vesicles and a large ER

B. Transport from Golgi to secretory vesicles

C. Normal number of vesicles and a small ER

D. Too few vesicles and a large ER

E. Excess vesicles and a small ER

D. Too few vesicles and a large ER
Reason: If vesicles can’t be formed in first place (sec12), then the defect in vesicle fusion (sec17) becomes irrelevant.

iClicker

The Sec42 mutant has an excess of secreatory vesicles near the plasma membrane compared to wild-type. In the sec22 mutant, proteins that are supposed to be secreted accumulate in the Golgi. What would you predict a sec22, sec42 double Mutant would look like?

A. Enlarged ER and too few vesicles

B. Secreted proteins accumulate in Golgi

C. Excess secretory vesicles near the plasma membrane

D. None of the above

B. Secreted proteins accumulate in Golgi

Reason: cytoplasm → ER → vesicle → Golgi → vesicle → outside the cell. Proteins will accumulate in Golgi (sec22) before making it to vesicles near pm (sec42)

Lecture 14 5/5

Synthesis of Secretory, Lysosomal, or plant vacuolar proteins

1. Co-translocation deposits protein into the ER lumen

2. Polypeptide signal sequence 6-15 hydrophobic amino acid resides, targeting pp to ER membrane

3. Signal sequence recognized by signal recognition particle (SRP)

4. SRP binds pp and ribosome, arresting synthesis

5. complex is recruited to ER thru interactions btwn SRP and SRP receptor

6. Ribosome goes to translocon. Once attached, signal sequence is recognized, pp is inserted into translocon channel

iClicker

Why is the band in lane 1 smaller than the band in lane 2?

A. The protein in lane 1 has a signal peptide

B. The protein in lane 2 has a signal peptide

C. The protein in lane 1 is a transmembrane protein

D. The protein in lane 2 is unfolded

B. The protein in lane 2 has a signal peptide

iClicker

Which protein would you suspect is secreted?

A. Protein 1 (black)

B. Protein 2 (red)

C. Both.

D. Neither one.

B. Protein 2 (red)

iClicker

If you add the nuclear localization sequence PKKKRKV to the beginning (N-terminus) of a secreted protein, where would you expect to find the protein?

A. The cytosol

B. The nucleus

C. The ER

D.Outside the cell

E. None of the above

D. Outside the cell

Reason: Secreted proteins already have an N-terminal ER signal sequence which directs the ribosome to the ER during translation. This signal overrides any downstream sequences like an NLS (PKKKRKV) and has no effect (following secretory pathway and ending up outside the cell)

iClicker

If you add the nuclear localization sequence PKKKRKV to the end (C-terminus) of an ER lumen protein, where would you expect to find the protein?

A. The cytosol

B. The nucleus

C. The ER

D.Outside the cell

E. None of the above

c. The ER

Reason: NLS must be in the cytoplasm to be recognized by importins that mediate nuclear import. Protein was already target to the ER lumen when NLS was added to C-terminus, so it stays bc no cytoplasm

iClicker

You have isolated a yeast strain with a mutation in the SRP. This mutant SRP can not bind nucleotides. Where would you predict to find a protein that is secreted?

A. Never made

B. Partially made, but still attached to ribosomes

C. In the cytoplasm

D. In the ER lumen

E. Secreted outside the cell

c. In the cytoplasm

Reason: SRP cannot bind, making it continue to translate in the cytoplasm and not be secreted.

Lecture 15 5/7

Type 1 membrane protein

• N-terminus in lumen (outside cell)

• single pass

• signal peptide + stop-transfer sequence

• C-terminus in cytoplasm

Type II membrane protein

• N-terminus in cytoplasm (inside cell)

• single pass

• C-terminus in lumen

• Internal signal-anchor sequence with (+) charged amino acids before internal sequence

Type III membrane protein

• N-terminus in ER lumen

• single pass

• C-terminus is in cytoplasm

• internal signal anchor sequence with (+) charged amino acids after internal sequence

Type 4 membrane protein

• Type IV-A: N-terminus in cytosol

• Type IV-B: N-terminus in lumen

• multi-pass

• Multiple internal start and stop sequences

• may or may not have signal peptide

• domains alternate btwn lumen and cytoplasm

• C-terminus in cytoplasm or lumen based on # of membrane spans

Hydrophobicity Plot of Type IV-B

• Protein has predicted signal sequence at its N-terminus (first peak) and 4 transmembrane spans (peaks after signal sequence)

Glycosylation in RER

1. Attachment of an Oligosaccharide (N-linked Glycosylation)

2. Catalyzed by oligosaccharyltransferase

3. Glucose and mannose residues are trimmed to prepare for further folding and processing

4. Glycan helps proper folding by interacting with chaperones like calnexin and calreticulin

5. Misfolded proteins will be glucose tagged, mannose deficient and degraded by proteasome (ER)

Mechanisms that Ensure Destruction of Misfolded Proteins (UPR)

• Accumulation of misfolded proteins triggers the unfolded protein response (UPR)

• Sensors in the ER are kept inactive by the chaperone BiP (BiP binds to unfolded proteins and sensors)

• When misfolded proteins accumulate, BiP is incapable of inhibiting sensors

• activated sensors send signals to trigger proteins in destruction of misfolded proteins

iClicker

Hypothesize why is the band in lane 1 smaller than the band in lane 2?

A. The protein in lane 2 is unfolded

B. The band in lane 1 has had its signal peptide cleaved

C. The protein encoded for by mRNA 1 is a multipass membrane protein.

D. The band in lane 2 has a signal peptide

E. Sugars were added to the protein in lane 2

E. Sugars were added to the protein in lane 2

Reason: Protein 2 has microsomes, which act like ER and allow glycosylation to happen. Sugars are added to the protein, making it larger so it runs slower on SDS-PAGE.

Lecture 16 05/09

How are materials carried between compartments

using coated vesicles

Protein coats 2 functions

1. Cause the membrane to curve and form a vesicle

2. Select the components to be carried by vesicle

Using cell-free assays to study membrane and secreted proteins (glycosylation)

• Lane 1

  • Only mRNA and the lysate

• Lane 2

  • mRNA + lysate + microsomes → L2 has a larger size than L1 bc L2 undergone glycosylation

• Lane 3

  • mRNA + lysate + microsomes + tunicamysin → Lane 3 is smaller than L2 and L1 bc tunicamycin will block glycosylation (aka sugars aren't added, and its smaller than Lane 1 bc the signal sequence was cleaved inside microsome)

• Lane 4

  • mRNA + lysate + microsomes + the protease + tunicamycin → L4 has the same band size as L3 bc inside the protein is already inside the microsomes. Addition of protease does not change the size of the protein

• Lane 5

  • mRNA + lysate + protease → Nothing on this gel bc w/o microsomes, the protein is left inside the cytoplasm to be digested by protease

• Lane 6

  • mRNA + lysate + microsomes + protease -> Same as L2 bc glycosylation is occurring, sugars are being added so it's heavier than L3 and L4

creds to mbau <3

Types of Vesicle Transport

• COPII-coated vesicles

• COPI-coated vesicles

• Cathrin-coated vesicles

COPII-coated vesicles

• moves proteins from the rough endoplasmic reticulum to the Golgi apparatus (anterograde transport) “forward”

1. Cargo Selection

   • Proteins defined for the Golgi are identified in the ER (ER exit sites)

2. Coat Protein Assembly (COPII Coat Formation)

   • Sar1 is activated by GTP and inserts into the ER membrane

   • Sar1 recruits COPII proteins: Sec 23, Sec24, Sec13, Sec31

     • Forms coat that bends membrane & helps form vesicle

3. Vesicle budding

   • Coated vesicle buds off from ER

4. Coat Disassembly

   • After budding, COPII coat is shed, required for docking at Golgi

5. Vesicle Targeting and Fusion

   • uncoated vesicle uses SNARE proteins to fuse w/cis-Golgi membrane

SNAREs (v & t)

• integral proteins that bring the vesicle and target compartment in close contact

• v-SNAREs are found in transport vesicles, pairs with t-SNAREs to initiate fusion

• t-SNAREs are found in target compartments that facilitate vesicle fusion in intracellular transport

t-SNARE mutant

• vesicle docking may occur but fusion fails

• Proteins and vesicles accumulate near target membrane

• specific effects depend on which t-SNARE is mutated

v-SNARE mutant

• vesicle fusion fails

• secretory proteins accumulate in vesicles

• Blocked secretion or disrupted organelle function

Rabs

• proteins that cycle btwn GTP and GDP

• GTP-bound Rabs associate w/membranes by a lipid anchor

COPII-uncoated vesicles

1. Vesicle Formation:

   • A COP II vesicle forms at the ER membrane, enclosing cargo proteins.  

2. Coat Removal:

   • The COP II coat disassembles, leaving an uncoated vesicle.  

3. Rab Activation:

   • Rab-GEF activates Rab-GDP to Rab-GTP on the vesicle.  

4. Tethering:

   • Rab-GTP on the vesicle interacts with a Rab effector on the cis-Golgi membrane, tethering the vesicle.  

5. SNARE Pairing:

   • v-SNAREs on the vesicle bind to t-SNAREs on the cis-Golgi membrane, forming a SNARE complex.  

6. Membrane Fusion:

   • The SNARE complex facilitates fusion of the vesicle membrane with the cis-Golgi membrane.  

7. Cargo Release:

   • The vesicle's contents are released into the cis-Golgi lumen

COP1 coated vesicles

• For transport from cis-Golgi to ER (retrograde); “backwards”

• Maintained by:

  1. Retention of resident molecules excluded from transport vesicles

  2. Retrieval of “escaped” molecules back to normal compartment

• Moves Golgi resident enzymes in trans-to-cis direction and ER resident enzymes from ERGIC & Golgi complex back to ER

Cathrin-coated vesicles

• Essential for selective transport btwn cellular membranes

  • move materials from the trans-Golgi network (TGN) to endoscopes, lysosomes, and plant vacuoles (plasma membrane)

  • Endocytosis from plasma membrane

Ran

• Is a GTPase that can be bound to GTP or GDP

• Ran-GEF act to create a gradient of Ran-GTP in the nucleus

• Ran-GAP acts to create a gradient of Ran-GDP in the cytoplasm

Practice Question

Match the statement with the coated protein that it describes.

[Clathrin, COPI, COPII, All of them, None of them]

1. Delivers molecules out of the ER
2. Uses Sar1
3. Uses PI(4,5)P2
4. Used in retrograde transport
5. Make buds that are referred to as pits
6. Can be recruited by coat-recruitment GTPases

7. Interacts with Rabs
8. Recognizes the KDEL sorting signal
9. The outer coat layer is composed of a protein with a triskelion structure of heavy and light chains.
10. The inner coat layer is composed of Sec23/24.
11. Auxilin aids in uncoating.
12. Recognizes cargo receptors with C-terminal KKXX motifs.

1. COPII
2. COPII
3. Clathrin
4. COPI
5. Clathrin
6. All of them
7. None of them
8. None of them
9. Clathrin
10. COPII
11. Clathrin
12. COPI

Practice Question

Select all statements that are TRUE about monomeric GTPases.
1. Rab is lipid-anchored when active.
2. Rab-GAP recruits Rab to the membrane.
3. Arf recruits to Sec13/31.
4. Rab-GTP is bound to GDI.
5. Sar1-GAP leads to vesicle uncoating.
6. Arf is associated with a membrane when it is bound to GTP.

1. Rab is lipid-anchored when active.
6. Arf is associated with a membrane when it is bound to GTP.

Practice Question

Which is false about co-translocation?

1. N-terminal signal sequences are cleaved off via a signal peptidase.
2. Start and stop transfer sequences are indistinguishable.
3. The signal sequence opens the seam of the translocator.
4. BiP helps pull the protein into the lumen of the ER.
5. SRP binds to the signal sequence and the ribosome to pause translation

4. BiP helps pull the protein into the lumen of the ER.

Practice Question

Indicate whether each description better applies to COPI (1), COPII (2), or clathrin (3) coated vesicles. your answer will be a 3 digit number composed of digits 1 to 3 only, eg. 123.

____ Involved in anterograde transport from the ER
____ Involved in transport from the plasma membrane
____ Involved in transport from Golgi to Golgi

2, 3, 1

Practice Question

#3 is a start- or stop- transfer sequence?

start