Intracellular Compartments and Protein Transport Notes

Intracellular Compartments and Protein Transport

Introduction

A typical eukaryotic cell performs thousands of chemical reactions simultaneously.
Many of these reactions are incompatible and must be segregated for effective cell operation.
Two main strategies for organizing these reactions:
- Aggregating enzymes into large multicomponent complexes (e.g., DNA, RNA, and protein synthesis).
- Confining metabolic processes and related proteins within membrane-enclosed compartments.

Membrane-Enclosed Compartments/Organelles

Eukaryotic cells utilize membrane-enclosed compartments, also known as organelles, to isolate and organize different chemical reactions.

Organelles and Their Functions

Nucleus: Enclosed by a double membrane (nuclear envelope) and communicates with the cytosol via nuclear pores.
Endoplasmic Reticulum (ER): Major site for the synthesis of new membranes.
- Rough ER: Contains ribosomes on its cytosolic surface, involved in protein synthesis and modification.
- Smooth ER: Lacks ribosomes, involved in lipid synthesis and other metabolic processes.
Golgi Apparatus: Receives proteins and lipids from the ER, modifies them, and dispatches them to other destinations.
Lysosomes: Contain digestive enzymes that degrade worn-out organelles, macromolecules, and particles taken into the cell by endocytosis.
Endosomes: Compartments involved in sorting and trafficking endocytosed materials.
Peroxisomes: Single-membrane organelles containing enzymes for oxidative reactions.
Mitochondria: Involved in pyruvate oxidation, the TCA cycle, and oxidative phosphorylation.
Chloroplasts: (In plant cells) Involved in photosynthesis.

Organelle Volume

The relative volumes of membrane-enclosed organelles vary depending on the cell type and function.

Evolution of Organelles

Nuclear membranes and ER: May have evolved through invagination of the plasma membrane in ancient prokaryotic cells, forming a double-layered envelope around the DNA.
Mitochondria: Thought to have originated when a prokaryote was engulfed by a larger eukaryotic cell.
Chloroplasts: Thought to have originated when a eukaryotic cell engulfed a photosynthetic prokaryote.
These theories explain why mitochondria and chloroplasts have two membranes and their own genomes.

Protein Sorting

Most protein synthesis begins in the cytosol.
Exceptions: few mitochondrial and chloroplast proteins.
Sorting signal: Directs a protein to the organelle where it is required.
Three main mechanisms for protein import into organelles:
1. Transport through nuclear pores: From the cytosol into the nucleus.
2. Transport across membranes: From the cytosol into the ER, mitochondria, or chloroplasts.
3. Transport by vesicles: From the lumen of one compartment of the endomembrane system to another.

Signal Sequences

Proteins destined for the ER possess an N-terminal signal sequence that directs them to that organelle.
Proteins destined to remain in the cytosol lack such a sequence.

Nuclear Pores

The double membrane of the nuclear envelope is penetrated by nuclear pores, facilitating bidirectional traffic.
Traffic includes newly made proteins, RNA molecules, and ribosomal subunits.
The outer nuclear membrane is continuous with the ER.

Nuclear Pore Complex

Nuclear pores contain binding sites for chromosomes and provide anchorage for the nuclear lamina.
They are water-filled passages containing many proteins with unstructured regions, preventing the passage of large molecules.

Nuclear Transport

Nuclear Localization Signal (NLS): Directs a protein from the cytosol into the nucleus.
Nuclear Transport Receptors: Bind to the NLS on newly synthesized proteins destined for the nucleus.

Energy-Driven Nuclear Transport

Nuclear transport receptors pick up cargo proteins in the cytosol and enter the nucleus.
In the nucleus, Ran-GTP binds to the nuclear transport receptor, causing it to release its cargo.
The nuclear transport receptor, still carrying Ran-GTP, is transported back to the cytosol.
In the cytosol, an accessory protein triggers Ran to hydrolyze its bound GTP to GDP.
Ran-GDP falls off the nuclear transport receptor, which can then bind another cargo protein destined for the nucleus.
The energy supplied by GTP hydrolysis drives nuclear transport.

Protein Import into Mitochondria

The mitochondrial signal sequence of a precursor protein is recognized by a receptor in the outer mitochondrial membrane.
The receptor-protein complex diffuses to a contact site, where the protein is translocated across both membranes by a protein translocator.
The signal sequence is cleaved off by a signal peptidase inside the mitochondrion.
Chaperone proteins help pull the protein across the membranes.
Proteins are imported into mitochondria in an unfolded form.

Endoplasmic Reticulum (ER)

The ER is the most extensive membrane network in eukaryotic cells.

Ribosomes and Protein Synthesis

Membrane-bound ribosomes: Attached to the cytosolic side of the ER membrane.
Free ribosomes: Unattached to any membrane.
A common pool of ribosomes is used to synthesize both proteins that stay in the cytosol and those that enter the ER.
At the end of each round of protein synthesis, ribosomal subunits are released to rejoin the common pool.

SRP and ER Targeting

The Signal-Recognition Particle (SRP) binds to the exposed ER signal sequence and to the ribosome, slowing protein synthesis.
The SRP-ribosome complex binds to an SRP receptor in the ER membrane.
SRP is released, passing the ribosome to a translocation channel in the ER membrane.
The translocation channel inserts the polypeptide chain into the membrane and starts transferring it across the lipid bilayer.

Protein Translocation into the ER Lumen

A translocation channel binds the signal sequence and actively transfers the polypeptide across the lipid bilayer as a loop.
During translocation, the signal peptide is cleaved by a signal peptidase.
The cleaved signal is ejected into the bilayer and degraded.
The translocated polypeptide is released as a soluble protein into the ER lumen.

Integration of Transmembrane Proteins

For single-pass transmembrane proteins, an N-terminal ER signal sequence initiates transfer.
The channel discharges the protein sideways into the lipid bilayer.
The N-terminal signal sequence is cleaved off, leaving the transmembrane protein anchored in the membrane.
Double-pass transmembrane proteins use an internal ER signal sequence as a start-transfer signal.
When a stop-transfer sequence enters the channel, both sequences are discharged into the membrane.
Neither sequence is cleaved, and the peptide remains anchored in the membrane.
Proteins spanning the membrane multiple times contain further pairs of stop and start sequences, repeating the process for each pair.

Recap: Intracellular Compartments and Transport

Eukaryotic membrane-enclosed organelles include the nucleus, ER, Golgi apparatus, and lysosomes.
Most organelle proteins are made in the cytosol and transported into the organelle.
Sorting signals guide proteins to the correct organelle; cytosolic proteins lack such signals.
Nuclear proteins contain nuclear localization signals (NLS) for import through nuclear pore complexes.
Mitochondrial proteins are made in the cytosol and must be unfolded to pass through protein translocators.
The ER is the membrane factory of the cell, synthesizing lipids and proteins.
Ribosomes are directed to the ER by an ER signal sequence recognized by a signal-recognition particle (SRP).
Soluble proteins pass completely into the ER lumen, while transmembrane proteins remain anchored by membrane-spanning α helices.

Vesicular Transport

Vesicular transport is highly organized between compartments of the endomembrane system.
The outward secretory pathway transports proteins from the ER through the Golgi to the plasma membrane or lysosomes.
The inward endocytic pathway ingests extracellular molecules in vesicles and delivers them to endosomes and lysosomes.

Clathrin-Coated Vesicles

Vesicles start as Clathrin-coated pits at the plasma membrane.
These vesicles bud from the plasma membrane on the endocytic pathway and from the Golgi on the secretory pathway.
Clathrin forms a basketlike cage that helps shape membranes into vesicles.

Cargo Selection and Vesicle Budding

Cargo receptors, with their bound cargo molecules, are captured by adaptins, which also bind Clathrin molecules.
Dynamin proteins assemble around the neck of budding vesicles, hydrolyze GTP, and pinch off the vesicle.
After budding, coating proteins are removed, and the vesicle can fuse with the target membrane.

Vesicle Targeting

A tethering protein on a membrane binds to a Rab protein on the surface of a vesicle.
A v-SNARE on the vesicle binds to a complementary t-SNARE on the target membrane.
Rab proteins and SNAREs direct transport vesicles to the target membrane.

Membrane Fusion

Pairing of v-SNAREs and t-SNAREs brings the two lipid bilayers into close proximity.
The force of the SNAREs winding together squeezes out water molecules, allowing lipids to flow together and form a continuous bilayer.
SNARE proteins play a central role in membrane fusion.

Secretory Pathways

Exocytosis: Newly made proteins, lipids, and carbohydrates are delivered from the ER, via the Golgi, to the cell surface by transport vesicles.
Glycosylation: Addition of oligosaccharide chains to the $NH_2$ region of an asparagine in the polypeptide.
These are called N-linked oligosaccharide chains.
Each oligosaccharide chain is transferred as an intact unit to the asparagine from a lipid, catalyzed by a membrane-bound oligosaccharide protein transferase with its active site exposed on the luminal side of the ER membrane.
Many proteins are glycosylated in the ER.

Protein Quality Control in the ER

Chaperones prevent misfolded or partially assembled proteins from leaving the ER.
Misfolded proteins in the ER lumen trigger the production of chaperones and the expansion of the ER, a system known as the unfolded protein response (UPR).
Binding to receptors that stimulate the production of a transcriptional regulator.
The protein translocates to the nucleus where it activates genes that encode chaperones and other ER components

Golgi Apparatus

Each Golgi stack has two distinct faces and consists of membrane-enclosed sacs (cisternae).
Soluble proteins and membrane enter the cis Golgi network via transport vesicles derived from the ER.
Proteins travel through the cisternae in sequence using transport vesicles that bud from one cisterna and fuse with the next.
Proteins exit the trans Golgi network via transport vesicles destined for either the cell surface or another compartment.

Constitutive and Regulated Exocytosis

Many soluble proteins are continually secreted from the cell by the constitutive secretory pathway, which operates in all cells.
This pathway also continually supplies the plasma membrane with newly synthesized lipids and proteins.
The regulated exocytosis pathway operates only in cells specialized for secretion.
Specialized secretory cells produce large quantities of particular products stored in secretory vesicles.
In secretory cells, the regulated and constitutive pathways of exocytosis diverge in the trans Golgi network.

Endocytic Pathways

Two main types of endocytosis:
- Pinocytosis (cellular drinking): Ingestion of fluid and molecules (small vesicles).
- Phagocytosis (cellular eating): Ingestion of large particles, such as microorganisms or cell debris (large vesicles called phagosomes).
Phagocytic cells ingest other cells.

Receptor-Mediated Endocytosis of LDL

Cholesterol is transported in the bloodstream bound to protein in the form of low-density lipoproteins (LDL).
LDL binds to receptors on the cell surface and are internalized in clathrin-coated vesicles.
The vesicles lose their coat and fuse with endosomes.
In the acidic environment of the endosome, LDL dissociates from its receptors.
LDL ends up in lysosomes, where it is degraded to release free cholesterol.
LDL receptors are returned to the plasma membrane via transport vesicles for reuse.

Fate of Receptor Proteins

The fate of receptor proteins involved in endocytosis depends on the type of receptor.

Lysosomes

A lysosome contains hydrolytic enzymes and an $H^+$ pump.
Materials destined for degradation follow different pathways to the lysosome.