chp. 15: intracellular compartments + protein transport

membrane-enclosed organelle

Any organelle in a eukaryotic cell that is surrounded by a lipid bilayer—for example, the endoplasmic reticulum, Golgi apparatus, and lysosome.

• prokaryotes dont have membrane-bound organelles

• create enclosed compartments that segregate different metabolic processes

cytosol

the gel-like, water-based fluid within cells—surrounding organelles

• cytoskeleton is here to keep organelles in place

nuclear envelope

the double membrane surrounding the nucleus, encloses the DNA and defines nuclear compartment

• Consists of outer and inner membranes, perforated by nuclear pores.

nuclear pores

penetrates the nuclear envelope of eukaryotic cells, acting as the sole, highly selective channel for transport between the nucleus and cytoplasm

• a large elaborate structure composed of 30 diff proteins, each present in multiple copies.

what is the outer nuclear membrane continuous with?

the endoplasmic reticulum

endoplasmic reticulum

is major site of synthesis of new membranes in the cell

• has rough and smooth ER

• continuous with nuclear envelope

• has a phospholipid bilayer membrane

ER interior = lumen

rough ER

ER with ribosomes attached to cystolic surface

• site of protein synthesis for secretion and membranes.

(remember: ribosomes make proteins)

smooth ER

ER without ribosomes

• involved in lipid synthesis, detoxification, and Ca²⁺ storage.

golgi apparatus

Modifies, sorts, and packages proteins and lipids for transport to other parts of the cell

• near the nucleus and receives the proteins + lipids from the ER

lysosomes

Digest and break down damaged organelles, cellular waste, and engulfed materials from endocytosis.

• before endocytosed materials go to lysosomes, they must pass the endosomes.

endosomes

Sort endocytosed material and recycle components back to plasma membrane

• they then send material to lysosomes for degradation

peroxisomes

small organelle that contains enzymes which break down lipids and destroy toxic molecules, producing H₂O₂ (hydrogen peroxide)

mitochondria

Produce ATP through oxidative phosphorylation.

chloroplast

Carry out photosynthesis (ATP production and carbon fixation).

Why do eukaryotic cells need internal membranes?

Large cell size requires compartmentalization to maintain efficiency and surface area-to-volume balance.

how are organelles positioned in the cell?

by attachment to the cytoskeleton, specifically microtubules

• they move organelles around to direct traffic

  • movement is driven by motor proteins that use energy from ATP hydrolysis to propel organelles/vesicles along filaments

How did membrane-bound organelles likely evolve?

From the expansion and infoldings (invaginations) of the plasma membrane.

How did early cells before eukaryotes organize membranes and how did internal membranes evolve?

Early cells (like ancient archaea and prokaryotes) only had a plasma membrane and it could carry out all membrane-dependent functions like ATP synthesis + lipid synthesis.

• this was fine cuz their small size gave them high SA:V ratio

how plasma membrane expansion made organelles

As cells grew larger, the plasma membrane expanded by having membrane protrustions, forming inward folds (invaginations) btwn these protrustions. These folds eventually pinched off and formed internal membrane-bound organelles like the ER, Golgi, and nuclear envelope.

endomembrane system

Interconnected network of membrane-enclosed organelles in a eukaryotic cell

• includes the endoplasmic reticulum, Golgi apparatus, lysosomes, peroxisomes, and endosomes.

How did the nuclear envelope likely form?

From membrane invaginations surrounding DNA that pinched off

What is the origin of mitochondria and chloroplasts?

Endosymbiosis—engulfed bacteria that evolved into organelles.

• they have their own DNA cuz they originated from bacteria that retained parts of their genomes.

• theyre NOT part of endomembrane system cuz they evolved from engulfed bacteria, not from membrane infoldings!!

How do organelles communicate in the endomembrane system?

by small vesicles that bud off of one organelle and fuse w/ another

• transport vesicles carry soluble cargo proteins, as well as the proteins and lipids that are part of the vesicle membrane, from one organelle to another.

In a typical human secretory cell, which of the following membranes has the largest surface area?

rough ER

• its folded up to form extensive maze

Where are most proteins synthesized?

On ribosomes in the cytosol.

What happens to proteins without a signal sequence?

They remain in the cytosol.

signal sequences

Amino acid sequence that directs a protein to a specific location in the cell, such as the nucleus or mitochondria.

• signal sequences are often removed from finished protein once it arrives at destination

  • exception is nuclear proteins that need it to re-enter the nucleus after nuclear envelope breakdown during cell division.

genetic engineering can change signal sequence

they can delete or transfer the signal sequence from one protein to another and it will change the destination of the protein

• ex: they removed signal from ER protein and moved it to cystolic protein, and both proteins switched places

Why is protein sorting necessary in eukaryotic cells?

Proteins must be delivered to the correct organelle for cell growth, division, and function.

• especially during division when organelles have to be duplicated, or growth requires new lipids for membrane

the 3 main protein transport mechanisms

1) nuclear pore complexes

• go into nucleus

2) transmembrane transport (protein translocators)

• enter mitochondria, chloroplast, ER

3) vesicle transport

• transport btwn ER and other endomembrane organelles

ALL REQUIRE ENERGY! protein remains folded for mechanism 1 and 3 but has to unfold for 2

Protein transport via nuclear pores

proteins enter cytosol → nucleus through nuclear pores

• nuclear pores penetrate both inner and outer nuclear envelope, and function like selective gates that actively transport specific macromolecules and allow free diffusion of smaller molecules.

protein transport across membranes (transmembrane transport)

these r proteins moving from the cytosol → ER, mitochondria, or chloroplasts

• they are transported across the organelle membrane by protein translocators located in the membrane

  • protein translocators require protein to unfold before it guides it across hydrophobic interior of membrane

vesicular protein transport

proteins moving on from ER → other part of endomembrane system are transported by vesicles

• they will pinch off from membrane of one compartment then fuse with the membrane of a second compartment.

  • deliver soluble cargo proteins, and proteins/lipids that r part of vesicle membrane

protein translocators

proteins moving from cytosol to ER, mitochondria, or chloroplast must be transported by this protein translocator

• located in the membrane

• requires protein to unfold before it guides it across hydrophobic interior of membrane

transport vesicles

the vesicles that transport proteins moving on from ER → other pt. of endomembrane system (golgi, ER, nuclear envelope, lysosomes, plasma membrane)

• they will pinch off from membrane of one compartment then fuse with the membrane of a second compartment.

  • deliver soluble cargo proteins, and proteins/lipids that r part of vesicle membrane

What is the key feature of signal sequences?

They are based on chemical properties (like hydrophobicity), not exact amino acid order.

• can be primary a.a sequence, or 3-dimensional structure formed by a.a. of the protein

inner nuclear membrane

inner part of nuclear envelope that contains proteins which act as binding sites for chromosomes, and provide anchorage for nuclear lamina.

outer nuclear membrane

is continuous with the rough endoplasmic reticulum and contains ribosomes

• resembles ER membrane

nuclear lamina

a finely woven meshwork of protein filaments that lines the inner surface of the inner nuclear membrane

• provides structural support for nuclear envelope

• shape is determined by nuclear lamina proteins!

disordered nuclear pore proteins

alot of the proteins lining the nuclear pore has large unstructured regions where the polypeptide chains r v disordered.

• these disordered segments form a soft, tangled meshwork that fills the center of the channel, preventing the passage of large molecules but allowing small, water-soluble molecules to pass freely and nonselectively between the nucleus and the cytosol.

perinuclear space

cavity btwn the nuclear inner and outer membrane

nuclear pore complex

giant complex (50-100 polypeptides)

• diameter of complex: 120 nm

• diameter of opening: 25 nm

where all protein import/export into nucleus happens

nuclear pore active transport

larger proteins and RNAs require a nuclear localization signal (NLS) in order to enter nucleus from cytosol

• requires energy

• NLS must be recognized by nuclear import receptors (importins)

nuclear localization signal (NLS)

larger proteins and RNAs must contain a nuclear localization signal (NLS) in order to enter nucleus from cytosol

• this is a 7 amino acid stretch of (+) charged a.a in the middle of the polypeptide

  • `lysines and arginines

• NLS will be recognized by nuclear import receptor protein!

nuclear import receptor protein (importins)

a cystolic protein receptor that recognize nuclear localization signal on proteins destined for nucleus.

• import/export is regulated by GTP

• guide the protein to a nuclear pore by interacting w nuclear pore fibrils

how does a nuclear import receptor (importin) enter nuclear pore with new protein?

Importin binds the protein’s NLS → moves to nuclear pore → passes through by interacting with pore proteins → releases protein when Ran-GTP binds in the nucleus.

• it interacts w fibrils extending from rim of pore in cytosol and grabs onto short repeated a.a sequences within the tangle of nuclear pore proteins that fill the center of the channel. Passes through by disrupting their interactions.

what happens when a nuclear pore is empty

short repeated a.a sequences that importin would normally bind to will instead bind to one another, forming a loosely packed gel.

What protein regulates nuclear transport direction?

Ran (a GTPase)

2 forms:

• Ran-GTP

  • high conc’n in nucleus

  • causes release of the cargo protein

• Ran-GDP

  • high conc’n in cytosol

  • GTP gone and GDP dissociates leaving importin free to pu another protein

Ran-GTP

a protein that regulates nuclear transport by binding GTP to the import receptor in nucleus → causes release of cargo protein in nucleus

• IN HIGH CONC’N IN NUCLEUS

• Ran-GEF → converts Ran-GDP → Ran-GTP (in nucleus)

• after releasing cargo, will go back to cytosol

Ran-GDP

a protein that regulates nuclear transport by binding GDP to the import receptor in cytosol → importin now back in cytosol will hydrolyze GTP into GDP. GDP will dissociate easily, so now receptor protein (importin) is free to pick up another protein destined for the nucleus.

• IN HIGH CONC’N IN CYTOSOL

• Ran-GAP → converts Ran-GTP → Ran-GDP (in cytosol)

What is the difference between Ran-GTP/Ran-GDP and Ran-GEF/Ran-GAP?

Ran-GTP and Ran-GDP are the two forms of the Ran protein (active vs inactive), while Ran-GEF and Ran-GAP are regulatory proteins that convert Ran between these forms (GEF adds GTP, GAP removes GTP).

• They are helper proteins that control Ran, NOT Ran

• The localization of these accessory proteins guarantees that the concentration of Ran-GTP is higher in the nucleus, thus driving the nuclear import cycle in the desired direction

Ran-GEF

Ran-GEF → converts Ran-GDP → Ran-GTP (in nucleus)

• accessory protein that helps Ran convert btwn GTP and GDP

Ran-GAP

Ran-GAP → converts Ran-GTP → Ran-GDP (in cytosol)

• accessory protein that helps Ran convert btwn GTP and GDP

What makes nuclear transport unique compared to other organelles?

Proteins enter fully folded through pores instead of being unfolded and threaded across membranes

Where are most mitochondrial and chloroplast proteins made?

even tho they have their own genomes, most of the proteins r made in the cytosol, then imported into organelle.

What directs proteins to mitochondria or chloroplasts?

An N-terminal signal sequence thats recognized by a receptor associated w/ translocator (TOM or TIM)

• signal sequence will be cleaved off by signal peptidase once it gets inside mitochondrial matrix

How many membranes must proteins cross to enter mitochondria/chloroplasts?

Two membranes (outer + inner).

• will happen at site where 2 membranes r close together so protein can cross both simultaneously

In what state are proteins transported into these chloroplast/mitochondria?

must be unfolded protein!!!

• TIM/TOM unfold protein in process of pulling it into mitochondria matrix

translocator of the outer membrane (TOM)

a protein complex that moves an unfolded protein across the outer membrane of a mitochondria into intermembrane space from cytosol

• binds to n-terminus of cytosol protein.

translocator of the inner membrane (TIM)

a protein complex that moves an unfolded protein across the intermembrane space into matrix of mitochondria

• binds to n-terminus of cytosol protein

What helps pull proteins into the mitochondria and refold them?

chaperone proteins which use ATP hydrolysis for energy

Where do peroxisomal proteins come from?

Mostly from the cytosol, some from the ER (via vesicles).

What signal directs proteins to peroxisomes and how r they transported in?

A short 3–amino acid signal sequence serves as an import signal

Then a cytosolic receptor binds the protein and escorts it inside.

What helps proteins cross the peroxisomal membrane?

a membrane translocator which is diff than the receptor cuz the receptor binds the protein and brings it to the organelle, while the membrane translocator helps move the protein across the membrane.

• translocator doesnt require protein to unfold like it does when its in mitochondria/chloroplast!

How can new peroxisomes form?

Vesicles from the ER can fuse with existing peroxisomes or mature into new ones.

What are the two types of proteins that enter the ER?

• Soluble proteins → enter ER lumen and can stay in lumen or r sent via vesicles to Golgi, lysosomes, or plasma membrane.

• Transmembrane proteins → embed in ER membrane

both of these will have an ER signal sequence (hydrophobic amino acids).

where does protein translation start?

all begins in cytoplasm

• then can be destined to stay in cytosol as free ribosome or sent to ER/Golgi

  • if sent to ER then translation occurs in rough ER

free ribosome

make proteins that stay in the cytosol, nucleus, chloroplast, mitochondria

what happens if ribosome is making protein with ER signal sequence?

Proteins with an ER signal sequence enter the ER and stay in the endomembrane system (ER → Golgi → lysosome/membrane/secretion), not the nucleus or mitochondria.

• translation will occur in the ribosomes on rough ER, and newly synthesized protein is translocated into lumen of ER (or transmembrane)

ER signal sequence

signal made up of 8+ hydrophobic amino acids that directs proteins to ER

• this signal sequence will be on amino terminus (N)

• holds open protein translocator

Do proteins leave the ER and go back to the cytosol?

No, they stay within the endomembrane system.

• They are sent via vesicles to Golgi, lysosomes, or plasma membrane.

  • soluble ER protein→ go into ER lumen → secreted or sent to endomembrane organelles

  • Transmembrane proteins → get embedded in ER membrane → stay in membranes as they move

how are membrane bound ribosomes and free ribosomes different?

they are structurally and functionally identical; they differ only in the proteins they are making at a particular time.

• are all part of a common pool

what r the 2 components that help guide ER signal sequences to ER membrane?

• signal recognition particle (SRP)

  • binds to ribosome and ER signal seq. to pause protein synthesis

• SRP Receptor

  • Binds SRP and brings the ribosome to the ER membrane.

Signal recognition particle (SRP)

protein that helps guide ER signal sequence into ER membrane by binding to the ER signal sequence + ribosome and slows synthesis of ribosome.

• when SRP binds to SRP receptor, it will be released for reuse while ribosome attaches to a protein translocator

• present in cytosol!

SRP Receptor

a protein that binds SRP and brings the ribosome to the ER membrane.

• found embedded in ER membrane

• when SRP binds to this receptor, SRP is released and the receptor will pass the ribosome to the protein translocator and protein synthesis starts again as it enters ER membrane

where is ER signal seq. located in most proteins?

at amino (N) terminus!

signal peptidase

a transmembrane enzyme in ER that cleaves off ER signal sequence

• signal sequence gets ejected and degraded after signal is done holding open the protein translocator so full protein passes into ER lumen

how does soluble protein cross ER membrane?

SRP receptor passes ribosome to protein translocator which binds the signal sequence and its threaded through this channel during translation.

• signal seq. holds open protein translocator

• signal peptidase then cuts off signal seq. and it gets degraded

• once C-terminus of soluble protein has passed through, protein is released into ER lumen

single pass transmembrane proteins

they use their N-terminal signal sequence to intitiate translocation into ER membrane

• this N-terminal signal will then be cleaved off

transfer is then stopped by a hydrophobic stop-transfer sequence

• stop-transfer seq. will STAY in bilayer and form alpha helical membrane

WILL HAVE: N-terminus on lumenal side of lipid bilayer and C-terminus on cystolic side

stop-transfer sequence

this is a seq. made of hydrophobic a.a on ER transmembrane proteins

• they will REMAIN in the bilayer in single and multipass proteins

• they will form an alpha helical membrane spanning segment that anchors protein in membrane

multipass transmembrane proteins

an ER transmembrane protein that uses a start-transfer sequence (instead of an N-terminal signal like single pass)

• still uses same stop- transfer sequence

  • NEITHER WILL BE REMOVED!

start-transfer sequence

this is a seq. made of hydrophobic a.a on ER transmembrane proteins

• it will NEVER be removed from polypeptide

• ONLY in multipass transmembrane proteins

What determines whether a protein is soluble or membrane-bound in the ER?

The presence of additional hydrophobic sequences (stop/start-transfer).

How do lipids reach other organelles?

can be delivered either by:

• vesicles (mainly within the endomembrane system like Golgi, lysosomes, plasma membrane) '

• by lipid-transfer proteins at membrane contact sites (especially for mitochondria and some other non-vesicular pathways).

remember lipids r made by smooth ER!

which organelle cannot receive proteins directly from cytosol?

Golgi!

• Proteins are delivered to the Golgi apparatus from the ER or from other components of the endomembrane system.

vesicular transport

Movement of material between organelles in the eukaryotic cell via membrane-enclosed vesicles

• typically goes ER → Golgi → other endomembrane organelle

• allows for exocytosis/endocytosis!

transport vesicles

membrane vesicles that bud from one membrane and fuse with another, carrying membrane components and soluble proteins between compartments of the endomembrane system and the plasma membrane.

• maintains orientation so cystolic side always faces cytosol and noncystolic side always faces lumen

endocytic vesicle pathway

Extracellular molecules are taken into vesicles from the plasma membrane → early endosome → late endosome → lysosome
- ingestion and degradation of extracellular molec.

vesicle budding is driven by assembly of protein coat

vesicles that bud from membranes have a distinctive protein coat on cystolic surface

• after budding off parent organelle, vesicle will shed its coat to allow its membrane to directly interact w/ membrane to which it will fuse

2 functions of coat:

• helps shape membrane into a bud

• it captures molecules for onward transport

clathrin-coated vesicles

clathrin is a protein making up coat of transport vesicle that buds from either the Golgi or plasma membrane!

• requires adaptor molecules that recognize cargo-specific proteins

  • cargo is specific!

vesicle secretory pathway

rough ER → transport vesicle → cis Golgi → medial Golgi → trans Golgi → transport vesicle → plasma membrane (some stay here) → outside of cell.

• can also send to endosomes then to lysosomes

• at trans Golgi it can split up into either

  • constitutive secretion

  • regulated secretion (special secretory cells that only release products when stimulated by external signal)

How does a clathrin-coated vesicle form ?

Cargo binds receptors → adaptins link receptors to clathrin → clathrin assembles into a coat that bends membrane into a pit → dynamin uses GTP to pinch off the vesicle → vesicle sheds coat → vesicle fuses with target membrane.

clathrin

A coat protein that forms a basket-like cage around budding vesicles in endocytosis and transport from the Golgi or plasma membrane

• adaptin attaches clathrin to vesicle membrane

What does dynamin do in vesicle formation?

A GTP-binding protein that forms a ring around the clathrin vesicle neck and pinches it off from the membrane using GTP hydrolysis.

What is the role of adaptins in clathrin-coated vesicles?

secure clathrin coat to vesicle membrane and help select specific cargo molecules for packaging into vesicles.

• a second class of coat protein

• secure specific cargo molec. by trapping the cargo receptors that bind them

What is vesicle docking and what does it depend on?

it is the specific attachment of a transport vesicle to its correct target membrane before fusion.

• requires SNAREs and tethers/Rab

Rab recognition → tethering → SNARE docking → membrane fusion.

What do Rab proteins do in vesicle targeting?

they’re monomeric GTPases that act as identity markers on vesicles that are recognized by tethering proteins on the target membrane.

• ensures vesicles fuse w/ correct membrane

tethering proteins

Proteins on the cystolic target membranes that bind Rab proteins on vesicles to capture and hold them near the correct destination.

• Rab and tethering proteins provide intial recognition btwn vesicle and target membrane

SNARE proteins

transmembrane proteins that mediate tight docking and fusion between vesicle and target membranes after tethering protein grabs Rab

2 types:

• v-snares = on vesicles

• t-snares = on target membranes

they also catalyze final fusion of 2 membranes

How do SNAREs cause membrane fusion?

membrane fusion is unfavorable cuz water must be displaced and lipid bilayers must come extremely close (~1.5 nm) before merging.

• However, the v-SNARE and t-SNARE wrap around each other rlly tightly which draws the lipid bilayers super close together and the winding together squeezes out any water trapped btwn the 2 membranes

  • allows fusion and contents r delivered