BETTER PROTEIN SORTING AND PRINCIPLES OF VESICULAR TRANSPORT

Endomembrane system

Includes the nucleus, endoplasmic reticulum, Golgi apparatus, lysosomes, endosomes, and plasma membrane—connected through vesicular transport

Non-endomembrane organelles

Include mitochondria, chloroplasts, and peroxisomes—import proteins directly from cytosol, not via vesicles

Protein sorting

Process by which newly made proteins are directed to their correct cellular destinations

Free ribosome proteins

Remain in cytosol or are imported post-translationally into nucleus, mitochondria, chloroplasts, or peroxisomes

Bound ribosome proteins

Ribosomes attach to rough ER, where translation continues and proteins enter ER lumen co-translationally

Proteins in RER lumen

May stay in ER, go to Golgi, lysosomes, plasma membrane, or be secreted from the cell via vesicular transport

Vesicular transport

Transport process that carries proteins and lipids between ER, Golgi, lysosomes, and plasma membrane using membrane-bound vesicles

Translocation

Post-translational import means protein is fully synthesized in cytosol before import

Co-translational import

occurs while the protein is being synthesized and threaded into the ER

Signal sequence

Short peptide tag acting like a “zip code” determining protein destination

Gated transport

Selective movement of folded proteins and RNA through the nuclear pore complex

Nuclear pore complex (NPC)

Large protein assembly spanning both nuclear membranes that regulates exchange of macromolecules between nucleus and cytoplasm

FG repeats

Phenylalanine-glycine sequences lining NPC channels that bind importins/exportins and guide cargo through pore

Importin

Receptor that binds cargo proteins containing an NLS and transports them through the NPC

Exportin

Receptor that binds NES-containing proteins and transports them from nucleus to cytoplasm

Nuclear localization signal (NLS)

Basic amino acid sequence (lysine/arginine-rich) directing proteins into the nucleus

Nuclear export signal (NES)

Leucine-rich sequence directing proteins out of the nucleus

Ran-GTP

Small GTPase bound to GTP in nucleus that drives cargo release and receptor recycling

Ran-GDP

Form of Ran found in cytosol after GTP hydrolysis

Ran-GAP

Cytosolic enzyme that hydrolyzes Ran-GTP to Ran-GDP

Ran-GEF

Nuclear enzyme that converts Ran-GDP back to Ran-GTP

Energy source for gated transport

Hydrolysis of GTP by Ran provides energy for directionality

Nuclear import Step 1

Cargo with NLS binds importin in cytosol

Nuclear import Step 2

Importin-cargo complex interacts with FG repeats and moves through NPC

Nuclear import Step 3

Run-GTP binds importin in nucleoplasm causing cargo release

Nuclear import Step 4

Importin-Ran-GTP exits nucleus through NPC

Nuclear import Step 5

Run-GAP in cytosol hydrolyzes GTP to GDP causing complex dissociation

Nuclear import Step 6

Run-GDP diffuses back into nucleus

Nuclear import Step 7

Run-GEF exchanges GDP for GTP restarting the cycle

Regulation of nuclear import

Phosphorylation or inhibitory proteins can hide NLS to prevent nuclear import until activation is needed

Mitochondrial import type

Post-translational import of unfolded precursor proteins synthesized in cytosol

Presequence

N-terminal amphipathic helix that directs proteins to mitochondria

TOM complex

Translocase of the outer mitochondrial membrane recognizing the presequence

TIM23 complex

Translocase of the inner mitochondrial membrane that transports precursor into matrix

Mitochondrial Hsp70

Matrix chaperone that uses ATP to pull protein into matrix and prevent backsliding

Matrix processing peptidase (MPP)

Cleaves presequence once the protein enters mitochondrial matrix

Energy for mitochondrial import

ATP hydrolysis and proton-motive force across inner membrane

Protein folding state (mitochondria)

Imported in an unfolded state maintained by cytosolic Hsp70 until inside matrix

Chloroplast import type

Post-translational import using TOC/TIC complexes and ATP/GTP hydrolysis

Transit peptide

N-terminal sequence that targets precursor proteins to chloroplasts

TOC complex

Translocase of outer chloroplast membrane recognizing the transit peptide

TIC complex

Translocase of inner chloroplast membrane that imports protein into stroma

Stromal Hsp70

ATP-dependent chaperone pulling proteins into stroma and aiding folding

Stromal processing peptidase (SPP)

Removes transit peptide after import into stroma

Energy for chloroplast import

ATP and GTP hydrolysis (TOC uses GTP, TIC uses ATP)

Membrane potential (chloroplast)

Only across thylakoid membrane, not across inner chloroplast envelope

Differences between mitochondria and chloroplast import

Mitochondria use proton-motive force + ATP, chloroplasts use ATP + GTP and lack inner membrane potential

Folding after import

Proteins fold inside matrix (mitochondria) or stroma (chloroplast)

Shared traits of mitochondria and chloroplast import

Both require cytosolic chaperones, double membranes, N-terminal targeting sequences, translocases, and energy consumption

Vesicle

Small spherical membrane-enclosed structure (40–100 nm) that transports cargo between organelles

Donor compartment

Organelle membrane from which a vesicle buds off

Target compartment

Organelle membrane a vesicle fuses with to deliver cargo

Soluble cargo proteins

Proteins carried inside the vesicle lumen

Membrane cargo proteins

Proteins embedded within the vesicle membrane

Vesicle budding

Process in which part of a donor membrane curves outward and pinches off to form a vesicle

Anterograde transport

Forward movement of vesicles carrying cargo from ER → Golgi → membrane or lysosomes

Retrograde transport

Reverse movement that recycles lipids, receptors, and coat proteins back to donor compartments

GEF (guanine nucleotide exchange factor)

Activates Sar1 by exchanging GDP for GTP on its binding site in the ER membrane

Sar1

Small GTP-binding protein that initiates COPII vesicle formation

Sar1-GTP

Active membrane-bound form of Sar1 that recruits COPII coat proteins

COPII coat proteins

Cytosolic coat proteins that shape the vesicle and select cargo for ER → Golgi transport

Cargo selection

COPII binds transmembrane cargo and receptors attached to soluble cargo in ER lumen

Budding process

COPII assembly curves the ER membrane outward forming a coated pit that pinches off as a coated vesicle

Coated pit

Pit-like indentation in ER lumen formed during budding covered by COPII on the cytosolic side

Coated vesicle

Fully formed COPII-covered sphere containing cargo, receptors, and fusion proteins

Vesicle uncoating

Sar1 hydrolyzes GTP→GDP causing Sar1 and COPII to detach from vesicle

Sar1 recycling

Sar1-GDP and COPII return to cytosol for reuse in new vesicle formation

Docking

First step of vesicle fusion where vesicle recognizes and attaches to target membrane

Rab-GTP

G-protein on vesicle that binds to a specific tethering protein on the target membrane

Tethering protein

Flexible protein on target membrane that connects with Rab-GTP to bring membranes close

Rab–tether specificity

Each Rab interacts with a specific tether ensuring vesicle–target accuracy

v-SNARE

Fusion protein on vesicle membrane

t-SNARE

Fusion protein on target membrane

SNARE pairing

v-SNAREs and t-SNAREs coil together forming a four-helix bundle that pulls membranes close

Membrane fusion

Outer leaflets fuse first, then inner leaflets, mixing vesicle and target contents

Rab-GTP hydrolysis

After fusion, Rab hydrolyzes GTP→GDP, detaches from tether and leaves membrane

Rab recycling

Rab-GDP returns to cytosol to be reused in future docking events

Energetics of fusion

Membranes must displace water to touch, process is energetically unfavorable and requires ATP indirectly

NSF (N-ethylmaleimide Sensitive Factor)

ATPase that powers SNARE separation after fusion

SNAP (Soluble NSF Attachment Protein)

Protein that links NSF to SNARE complex

NSF–SNAP complex

Positions around coiled v-/t-SNARE bundle and uses ATP hydrolysis to unwind them

SNARE resetting

ATP hydrolysis untwists SNAREs so they can participate in another fusion event

v-SNARE recycling

v-SNAREs travel back to donor membrane via new vesicles

t-SNARE readiness

t-SNAREs remain in target membrane ready for next vesicle

ATP usage in vesicle fusion

ATP is used to separate and reset SNAREs, not to drive membrane fusion itself

Docking vs Fusion

Docking uses Rab–tether recognition fusion uses SNARE pairing to merge membranes

Directionality of transport

Cargo proteins move one way membranes and machinery are recycled in both directions

Specificity of trafficking

Determined by unique Rab–tether pairs and SNARE combinations

Energy cycle summary

GTP drives Sar1 and Rab cycles, ATP resets SNAREs after fusion

Outcome of fusion

Vesicle contents merge with target lumen, vesicle membrane merges with target membrane