Cell Bio Exam 3

What Is the Endomembrane System?

Group of organelles that have membranes associates with eachother and proteins move via vesicles

What organelles are in the endomembrane system? what are not?

nuclear membrane, ER, Golgi, lysosomes, endosomes, and peroxisomes. excluded mitochondria and chloroplasts

What does it mean for protein delivery if an organelle is in the endomembrane system?

If included proteins eneter ER then are moved by vesicles to reach Golgi, lysosome, membrane or are secreted. 

Why aren’t Mitochondria and chloroplasts Part of the Endomembrane System? (Endosymbiont Theory)

Mitochondria and chloroplasts were once prokaryotes that were engulfed by eukaryotes
We know this because they have:

• Two membranes (original and plasma)

• Circular DNA (seen in prokaryotes)

• Divide by binary fission

• Prokaryotic-like ribosomes

• Cardiolipin in membranes (seen in prokaryotes) 

Therefore, they evolved separately and use their own import machinery

What does it mean for protein delivery if an organelle is not in the endomembrane system?

They require a different system to import proteins besides vesicles. Nuclear proteins: enter through nuclear pore, Mitochondrial proteins: enter via TOM/TIM channels

what is a signal sequence?

direct protein to correct location and typically cleaved off once protein reaches destination.

what must a signal sequence be?

nessacary (needed to get to correcy location) and sufficent (correct location)

what is a signal patch?

signal sequence that is only active when folded

what is the nuclear pore complex and why is it needed

• gate through which molecules enter and exit the nucleus

• needed to tightly regulate what comes in because DNA is in nucleus

What is the nuclear localization signal (NLS)

• A signal that proteins bound for the nucleus have

• formed by lysine/arginine positive charge

• forms a signal patch (created in a folded protein)

Is the NLS removed?

No, needed repeatly during cells life

are nuclear import proteins folded or unfolded?

folded because of the signal patch

helper proteins of nuclear import? (4)

• Importin (Nuclear Transport Receptor)

  • Binds NLS in the cytoplasm for import

  • moves cargo through nuclear pore

• Nuclear Pore Complex (NPC)

  • Allows import of large proteins only when bound to importin

• Ran-GTP (inside nucleus)

  • Binds importin → forces cargo release

• Ran-GDP (in cytosol)

  • Form created after GTP hydrolysis

  • Cannot remain bound to importin

what is the energy soucre of nuclear import

GTP hydrolysis by Ran drives directionality.

steps of nuclear import

• importin binds to NLS on folded cargo protein in cytosol

• Importin undergoes conformational change, so it is recognized by fibrils

• Receptors direct cargo protein to the nuclear pore by interacting with fibrils

• compound goes into the pore complex

• Inside the nucleus, Ran GTP binds to importin

• importing releases cargo protein back to the cytoplasm

• Importin and Ran GTP exit the nucleus

• Ran hydrolases GTP to GDP in cytosolthe

• Ran falls off and releases importin and repeats processthe

what id during nuclear import

• nls is mutated

• defective importin

• Ran GTP/GDP cycle fails

• cargo stays in cytosol

• no transport

• import stops because cargo cant unload and recpetor cant recyle

Where are nuclear proteins made?

On free cytosolic ribosomes.

Where are mitochondrial proteins made?

On cytosolic ribosomes.

Signal Sequence for mitochondria

• needed

• positive charges (argiine/lysine)

• N terminus

when protein enter mitochrondia are they folded or unfolded?

unfolded (linear) because pores are narrow, chaperones help keep unfolded

Helper Proteins for mitchondrial import? (5)

• Receptor protein on outer membrane — recognizes the signal.

• TOM complex (Translocase of Outer Membrane)

  • First channel protein encounters.

• TIM complex (Translocase of Inner Membrane)

  • Pulls protein into matrix.

• Chaperones 

  • Keep the protein unfolded

  • Pull the protein inside

• Signal Peptidase

  • Removes the N-terminal signal once inside

Energy Source of mitchondrial import

ATP for TIM

Is the signal cleaved in mitochondrial import?

Yes removed by signal peptidase

steps for mitchondrial import?

• signal recognized by the repressor on the outer membrane

• Translocases of the outer membrane (TOM) get protein out of the outer membrane

• TIM (translocase of the inner membrane) gets protein out of the inner membrane to the matrix, which needs ATP

• When Tom finds Tim, the protein goes through the channel

• Once the matrix protein is folded and the signal sequence is cleaved

effects of possible defects

1- Mutated signal sequence

2- Defective TOM/TIM

3- Chaperone failure

1- can’t bind the receptor, so it stays in the cytosol

2- protein stuck between membranes

3- protein folds and can’t enter

What is the significane of ER import

entry into endomembrane system, ER → Golgi → lysosome → endosomes → plasma membrane → secretion.

where do ER bound proteins start?

On free cytosolic ribosomes
When signal appears, ribosome is moved to the ER to finsih translation

ER signal sequence properties

• nonpolar hydrophic amino acis

• near N terminus

are proteins folded or unfolded during er import?

• Imported as a linear chain during translation
  - Folding happens INSIDE the ER lumen

helper proteins for ER import (5)

• SRP (Signal Recognition Particle)

  • Binds ER signal

  • Pauses translation

• SRP Receptor

  • Located in ER membrane

  • Accepts SRP+ribosome complex

• Translocator Channel (Sec61)

  • Opens to allow polypeptide to enter the lumen

• Signal Peptidase

  • Cuts signal off for soluble proteins

• ER Chaperones

  • Help folding inside ER

  • Prevent misfolding/unfolded protein response

energy source for ER import

GTP

is signal cleaved for er import

• YES → for soluble ER proteins

• NO → for many transmembrane proteins (internal start-transfer sequences stay embedded)

er import steps

• Ribosomes begin translation in the cytosol.

• ER signal emerges, the SRP binds and pauses translation

• SRP brings the ribosome to the SRP receptor on the ER

• ribsomeones transferred to the tranlocator

• SRP leaves and the polypeptide chain is threaded into the lumen via the translocation channel, and translation continues in the ER lumen

• Signal may be cleaved by signal peptidase.

• Protein folds with the help of ER chaperones.

• Post-translational modifications occur:

  • N-linked glycosylation (on Asn)

  • Disulfide bond formation (unique to ER lumen)

What happens if these are defective inthe ER import

• No SRP

• Translocator broken

• Signal peptidase missing

• Chaperone failure

• The protein continues in the cytosol, reaching the wrong location

• Proteins cant enter ER

• signal not removed, protein misfolded

• unfolded protein response ER stress

single pass (N terminus in lumen)

• N-terminal signal starts transfer

• Stop-transfer anchors protein

• Signal is cleaved

• Result:

  • N inside lumen

  • C in cytosol

  • transmembrane domain embedded

single pass (cytoplasmic N terminus)

• Internal start-transfer

• Not cleaved

• Result:

  • N in cytosol

  • C in lumen

  • transmembrane domain embedded

double pass/ more

• N and C terminus in cytosol

• Repeated start-transfer and stop-transfer sequences create multiple membrane spans.

• Orientation depends on the first start-transfer.

• Intenral start-transfer signal sequence never removed

Where does glycosylation occur?

ER lumen (N-linked - lumen side) → modified in Golgi

• sugar covalenly bonded = glycoprtiens

• occurs as protein is being translated and translocated

Disulfided bond formation

• occurs in ER lumen (oxidizing) between cysteine amino acid

• protects teritrary structure

what is direction of secretory (exocytic) pathway?

out of cell: goes outward from cell interior :ER → cis-Golgi → trans-Golgi → plasma membrane → secretion

purpose of exocytosis

• bring proteins to the cell membrane

• let’s specialized cells respond to the environment

• allows proteins to communicate with proteins outside of cell

• cell replaces membrane every 30 mins

purpose of endocytic pathway?

• Bring nutrients inside

• Remove old receptors

• Balance membrane added by exocytosis: fulid balance

• defend against pathogens

how do vesicles form?

budding

three basic steps of budding process

• picking the correct cargo

• building a coat around the budding vesicle

• pinch off the vesicle

how does the vesicle pick the correct cargo

Cargo doesn’t randomly fall into vesicles — it’s selected.

• Cargo receptors grab specific soluble proteins

• Adaptins connect these receptors to coat proteins
  

why does the cell build a coat around the budding vesicle? what are the three main types?

Coat proteins give the vesicle curvature and identity.

• clathrin, COPII, COPI

what do cargo recpetors do

bind specific proteins via signal

what does adaptins do

connect cargo recpetors to coat proteins

players or clathrin-coated vesicles?

• cargo receptors (bind soluble cargo)

• Adaptin (links receptors to clathrin)

• Clathrin (forms outer coat)

• Dynamin (GTPase that pinches off vesicle)
  

pathway of clathrin coated vesicle

Golgi → endosome; PM → endosome)

pathway of COP coated vesicle (1 and 2)

1: Golgi → ER or Golgi → Golgi

2: ER → cis-Golgi

what does COPII coated vesicles do?

transport from ER to cis golgi

COPII coated vesicles players

• Sar1 (GTPase)

  • GDP form = inactive in cytosol

  • GTP form = inserts into ER membrane

• Sec23/24 = inner coat + selects cargo

• Sec13/31 = outer coat

• No dynamin required

how do vesicles know where to go (identity)?

every vesicle has a molecular ID code so it fuses with the correct membrane.

Purpose of Vesicle Budding

• Move proteins between endomembrane organelles

• Deliver membrane components

• Select cargo with high specificity

• Maintain flow between ER → Golgi → PM

• Feed lysosomes during endocytosis

Rab proteins

• Small GTP-binding proteins

• Bound to vesicle membranes

• Each membrane type has a unique Rab

Tethering proteins

• On target membrane

• Recognize correct Rab

• Bring vesicle VERY close (~1.5 nm)

SNAREs

• v-SNAREs on vesicle

• t-SNAREs on target

• Form a 4-helix bundle to “zipper” membranes together

• Squeeze out water → allow membrane fusion

mechanism for vesicles fusing with target membrane

• Vesicle arrives at destination using microtubules + motor proteins

• Rab–tether interaction docks vesicle

• v-SNARE + t-SNARE wrap tightly, pulling membranes very close

• Water is forced out → membranes fuse

• Vesicle collapses into target membrane

• NSF + SNAP use ATP to separate SNAREs for reuse 

is vesicle fusion engertically favorable?

no. Fusion is energetically UNFAVORABLE because two membranes must come extremely close.

whta if Rab mismatch with tether

vesicle docks at wrong organelle or fails to dock → NO fusion.

what if v snare is mutated

Docking happens, fusion cannot occur → vesicle accumulates.

what if clathrin is missing

Receptor-mediated endocytosis fails

what if sar1 cant bind to GTP?

COPII coat cannot form → proteins trapped in ER.

what is Unfolded Protein Response (UPR)

UPR is a protective mechanism activated when misfolded proteins build up in the ER and there are not enough chaperones. Er is clogged bc chaperones are overwhelmed, and quality control can’t keep up.

what happens during UPR

Cell makes more chpaerone to fix misfolded proteins, protein synthesis slows down, er grows to give more space for protein folding.

why do cells need UPR

To prevent the ER from being damaged by misfolded proteins.
Without UPR, misfolded proteins would:

• clog the ER

• create toxic aggregates

• disrupt the entire endomembrane system

Constitutive exocytosis

• occurs in all cells all the time

  • unregulated

• supplies newly made lipids and proteins to the plasma membrane

• carries secreted proteins which are released to the outside of cell

what does Exocytosis deliver

• transmembrane proteins to the plasma membrane in correct orentations

• realses solu proteins to the extracellular environment

• new phospolipids

regulated secretion

• Specialized secretory cells

• Stored in vesicles wating for signal

• Released only when triggered

• Example: insulin release

endocytosis pathway

PM → vesicle → early endosome → late endosome → lysosome

3 types of endocytosis

pinocytosis, phagocytosis, Receptor-Mediated Endocytosis

Pinocytosis

“cellular drinking”

• constituve (always on)

• ingest parts of membrane w fluid that has small particles in it

• balances fluid loss in exocytosis

• Feeds lysosomes

why is lysosome unique?

enzymes only function inside lysosme due to low pH

receptor mediated endocytosis

• Specific cargo injesyed

• Requires clathrin (receptor selects specific cargo)

• Vesicles are destined for the lysosome

phagocytsois

• Large particles (bacteria, debris)

• Performed by specialized cells (immune system)

• Defense: neutrophils/macrophages kill pathogens

where does core-oligosaccharide modification occur

er

where is Mannose-6-phosphate (M6P) tag added, what does it do

cis golgi. Tells the cell: “This protein belongs in the lysosome”

how is mgp recongized

• M6P receptor (M6PR) binds M6P-tagged hydrolases

• M6PR packages them into clathrin-coated vesicles headed to endosome → lysosome

what pathway do lysosomal hydrolases follow?

ER → cis-Golgi → trans-Golgi → endosome → lysosome 

steps of lysosome pathway

• core oligosacchride added in er to hydrolase

• enters cis golgi and m6p adds

• m6p receptor recongized it in transgolgi

• vesicle buds off and enters endosome

• then enetrs lysomes and m6p and m6p receptor recyled

what is chondrocyte and purpose

• Only cell type in cartilage

• Produce & maintain extracellular cartilaginous matrix: allows joins to move

• Cartilage cushions joints, shapes bones & facial features

what is Fibroblasts and purpose

• Connective tissue cells sampled in Ana’s test

• Used in lab assays because they contain lysosomes

Why do LSDs affect cartilage?

• Chondrocytes get swollen, stop making matrix

• Cartilage becomes thin, weak, underdeveloped

• explains Ana’s:

  • Hip/knee stiffness

  • Club feet

  • Facial abnormalities

  • Reduced mobility

Why do Ana’s lysosomal hydrolases show up in her BLOOD?

Her blood tests showed extra hydrolase activity, and her fibroblasts had low activity (lysosomes empty

This means:

• Enzymes never reached the lysosome

• Enzymes were secreted outside the cell instead

• This only happens when the M6P tag is missing OR M6P receptor can’t bind (only one enzyme tags them so if it doesnt work none are tagged)

what happens if M6P tag is missing

the Golgi thinks hydrolases are just normal secreted proteins → sends them out of the cell → they end up in the bloodstream.

What went wrong in Ana? 

mutation in the enzyme(GNPTAB) that adds M6P tag on hydrolase.

• Without M6P → NOTHING gets to the lysosome

• EVERYTHING gets secreted

be familar w this

What are Intermediate Filaments?

• One of the three cytoskeletal filaments (MT, Actin, IF)

• Most stable filament: tp take on stress

• Rope-like → stretchy + strong

• Found mostly in animal cells under mechanical stress:

  • epithelial cells

  • neurons

  • muscle cells

IF monomer Structure

• One polypeptide

• Has head (N) and tail (C)

• Middle = α-helical rod

IF dimer Structure

• Two monomers twist together

• “Coiled-coil”

• Held by hydrophobic interactions

IF Tetramer structure

• Two dimers anti-parallel (opposite directions)

• Ends look the same → NO polarity

Final IF strucute

• 8 tetramers pack together

• Twist into a rope-like cable

• Rope can stretch → gives strength

• Won’t break easily

Why can IFs withstand mechanical stress?

• Rope-like structure distributes tension

• Can shift from α-helix to β-sheet when stretched

• Accessory proteins help cross-link them

Cytoplasmic IFs (3 types)

Keratin Filaments (epithelial cells)

Vimentin Filaments (connective tissue)

Neurofilaments (neurons)

Keratin Filaments (epithelial cells)

• Most diverse IF family (54 genes)

• Found in skin, hair, nails

• Give tensile strength

• Link to other cells through desmosomes

• Disease: epidermolysis bullosa simplex

  • Mutated keratin → skin blisters from minor friction

Vimentin Filaments (connective tissue)

• Found in bone, muscle cell

• Job = anchor organelles (nucleus, mitochondria, ER)

Neurofilaments (neurons)

• Found in axon cytoplasm

• Provide space-filling properties → increase axon diameter

Nuclear IFs (Lamins)

• Form nuclear lamina under nuclear envelope

• Provide nuclear shape + stability

• Regulate cell division:

  • Phosphorylate → disassemble in early mitosis

  • Dephosphorylate → reassemble in late mitosis

Which cells contain which IFs?

• Epithelial cells → Keratin

• Muscle cells → Vimentin-like IFs

• Neurons → Neurofilaments

• All animal cells → Nuclear Lamins