Intracellular Compartments and Protein Transport Study Guide

Overview of Intracellular Compartments and Protein Transport

• Conceptual Framework: Eukaryotic cells are subdivided into membrane-enclosed organelles that allow for compartmentalization of metabolic processes.
• Key Organelles and their Primary Functions (Table 15-1):     * Cytosol: Contains metabolic pathways (Chapters 3 and 13), protein synthesis (Chapter 7), and the cytoskeleton (Chapter 17).     * Nucleus: Contains the main genome; site of DNA and RNA synthesis.     * Endoplasmic Reticulum (ER): Synthesis of most lipids and synthesis of proteins for distribution to organelles or the plasma membrane.     * Golgi Apparatus: Modification, sorting, and packaging of proteins and lipids for secretion or delivery to other organelles.     * Lysosomes: Intracellular degradation.     * Endosomes: Sorting of endocytosed material.     * Mitochondria: ATP synthesis via oxidative phosphorylation.     * Chloroplasts (Plants): ATP synthesis and carbon fixation via photosynthesis.     * Peroxisomes: Oxidative breakdown of toxic molecules.
• Quantitative Data for a Typical Liver Cell (Hepatocyte - Table 15-2):     * Cytosol: $54\%$ of total cell volume; $1$ per cell.     * Mitochondria: $22\%$ of total cell volume; approx. $1700$ per cell.     * ER: $12\%$ of total cell volume; $1$ per cell.     * Nucleus: $6\%$ of total cell volume; $1$ per cell.     * Golgi Apparatus: $3\%$ of total cell volume; $1$ per cell.     * Peroxisomes: $1\%$ of total cell volume; approx. $400$ per cell.     * Lysosomes: $1\%$ of total cell volume; approx. $300$ per cell.     * Endosomes: $1\%$ of total cell volume; approx. $200$ per cell.
• Structural Significance:     * Membrane-enclosed organelles occupy ~$50\%$ of the total cell volume.     * The total membrane area of organelles can be $20$ to $30$ times greater than the area of the plasma membrane.     * The Endomembrane System comprises the nuclear envelope, ER, Golgi apparatus, lysosomes, vesicles, endosomes, and plasma membrane. Mitochondria are NOT part of the endomembrane system.

Importance of Compartmentalization

• Maintenance of Proximity: Keeps interacting molecules close together (e.g., ribosomes, electron transport complexes).
• Pathway Confinement: Restricts specific metabolic pathways to defined regions.
• Biomolecular Aggregates: Non-membrane enclosed structures like nucleosomes also contribute to organization.

Protein Sorting and Signal Sequences

• Three Methods of Protein Import:     1. Through Nuclear Pores: Transport from cytosol to nucleus.     2. Through Membrane Directly: Requires protein translocators; proteins usually must be unfolded (exception: peroxisomes).     3. Through Vesicle Fusion: Transport between compartments of the endomembrane system.     * Energy Requirement: All three methods require energy to function.
• Signal Sequences (Table 15-3):     * Direct proteins to specific destinations.     * Are generally removed after the protein reaches its destination.     * Import into ER: $+H_3N\text{-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln-}$ (Hydrophobic residues green).     * Retention in ER Lumen: $\text{-Lys-Asp-Glu-Leu-COO-}$ (KDEL sequence).     * Import into Mitochondria: $+H_3N\text{-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-}$ (Positively charged red).     * Import into Nucleus: $\text{-Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-}$.     * Export from Nucleus: $\text{-Met-Glu-Glu-Leu-Ser-Gln-Ala-Leu-Ala-Ser-Ser-Phe-Leu-Leu-}$.     * Import into Peroxisomes: $\text{-Ser-Lys-Leu-}$.

Transport into the Nucleus

• Structural Context: The nuclear envelope is a double membrane. The outer membrane is continuous with the ER membrane. Nuclear pores penetrate both membranes.
• Import Mechanism:     * Nuclear Localization Signal (NLS): Recognized by nuclear import receptors.     * Receptors interact with cytosolic fibrils to navigate the pore.     * Receptors return to the cytoplasm to be reused.
• Export Mechanism: Similar transport receptors operate in reverse (e.g., for mRNA export).
• Ran GTPase Cycle:     * Ensures unidirectional transport.     * Ran-GTP: High-energy form found in the nucleus; binds to the receptor, causing it to release its cargo.     * Ran-GDP: Low-energy form; occurs when GTP is hydrolyzed. The receptor dissociates from Ran-GDP in the cytosol.     * Receptors prefer Ran-GTP over cargo for interaction.

Transport into Mitochondria and Peroxisomes

• Mitochondrial Import:     * Proteins are translated in the cytosol and must be kept unfolded for transport.     * TIM and TOM Complexes: Protein translocators in the inner and outer membranes, respectively.     * Signal sequences are removed once the protein enters the matrix.
• Peroxisome Import:     * Functions: Breakdown of toxins, lipids, and alcohol; generation of hydrogen peroxide ($H_2O_2$); synthesis of phospholipids for the nervous system.     * Method 1: Direct transport from cytosol via receptors. Unlike mitochondria, folded proteins can be transported across the peroxisomal membrane.     * Method 2: Vesicle fusion from the ER.     * Zellweger Syndrome: Autosomal recessive disorder of peroxisome biogenesis. Symptoms include hypotonia, seizures, hepatomegaly, and early death.

The Endoplasmic Reticulum (ER)

• The most extensive membrane network in eukaryotes.
• Rough ER: Ribosome-associated; proteins enter the ER while being translated (co-translational translocation).
• Smooth ER: Involved in fat and steroid metabolism.
• Ribosomes: A common pool is used. Ribosomes are not permanently attached to the ER. The same ribosome can translate mitochondrial and ER proteins at different times.
• ER Targeting Mechanism:     1. Signal Recognition Particle (SRP): Binds the ER signal sequence and the ribosome, slowing translation.     2. SRP Receptor: Located in the ER membrane; acts as a "molecular matchmaker" to bring the ribosome to the translocator.
• Soluble Protein Import:     * The signal peptide is cleaved by signal peptidase.     * The protein is released into the lumen and refolds.
• Transmembrane Protein Import:     * Single-pass: Contains an N-terminal ER signal sequence and a hydrophobic stop-transfer sequence. The signal sequence is cleaved, and the stop-transfer sequence anchors the protein in the bilayer.     * Double-pass: Contains an internal start-transfer sequence (not cleaved) and a stop-transfer sequence (not cleaved). Both remain in the membrane.     * Multi-pass: Complex topologies are determined by multiple start and stop sequences.

Vesicular Transport: Budding and Fusion

• Three Stages: Budding, Travel, Fusion.
• 1. Budding:     * Coating: Vesicles are coated with proteins like clathrin, COPI, or COPII. Clathrin forms basket-like cages.     * Adaptins: Secure the clathrin coat to the vesicle membrane and help select cargo molecules.     * Dynamin: A GTPase that constricts the neck of the budding vesicle to "pinch" it off.     * Coats disassemble after the vesicle is released.
• 2. Travel:     * Vesicles travel along microtubules (cytoskeletal "highways").
• 3. Fusion:     * Rab Proteins: Small GTPases (approx. $60$ types) on the vesicle surface provide specificity.     * Tethering Proteins: On the target membrane, bind to specific Rab proteins.     * SNAREs: v-SNAREs (vesicle) and t-SNAREs (target) pair tightly, docking the vesicle and pulling bilayers together to force fusion.

The Secretory Pathway (Exocytosis)

• Protein Modifications:     * Disulfide Bonds: Formed between Cysteines in the ER to stabilize tertiary structure and protect against degradation.     * Glycosylation: Addition of sugar chains (N-linked if on Asparagine). Starts in the ER via Oligosaccharyl Transferase. Signal: Asp-X-Ser or Asp-X-Thr.
• ER Quality Control:     * Chaperones: Hold misfolded proteins in the ER until they fold correctly.     * Cystic Fibrosis: A mutation in the CFTR channel prevents it from folding correctly; it is retained in the ER and degraded, even if it could function at the surface.     * Unfolded Protein Response (UPR): Triggered by accumulation of misfolded proteins. Signals the expansion of the ER, increased chaperone production, and slowing of protein synthesis. Can trigger apoptosis (cell death) if the problem persists (e.g., related to Type II Diabetes).
• Golgi Apparatus:     * Structure: Consists of layers called cisterna. Cis face is the entry (towards ER); trans face is the exit (towards plasma membrane).     * Sorting: Proteins are sorted via vesicles or cisternal maturation. Further glycosylation occurs here.
• Exocytosis Pathways:     1. Constitutive: Unregulated; replenishes lipids/proteins and secretes soluble proteins in all cells.     2. Regulated: In specialized cells; requires a signal (e.g., hormone or neurotransmitter) for vesicle fusion. Proteins often aggregate at the low pH of the trans Golgi.

The Endocytic Pathway (Endocytosis)

• Types of Endocytosis:     1. Pinocytosis ("Cell Drinking"): Uptake of fluid/small molecules in small vesicles ($< 150\,nm$). Constitutive/unregulated.     2. Phagocytosis ("Cell Eating"): Ingestion of large particles/cells ($> 250\,nm$) via phagosomes. Performed by specialized cells like neutrophils, macrophages, monocytes, and microglia.     3. Receptor-mediated Endocytosis: Targeted uptake of specific macromolecules (e.g., LDL/cholesterol). Efficient and regulated.
• Low-density Lipoprotein (LDL) Pathway:     * LDL binds to LDL receptors -> clathrin-coated vesicles -> fusion with endosomes.     * Acidic pH in endosomes causes LDL to release from the receptor.     * Receptors are recycled; LDL goes to lysosomes to release free cholesterol.     * Familial Hypercholesterolemia: Mutation in LDL receptor leads to high blood cholesterol and yellow plaques around eyes.
• Autophagy ("Self-Eating"): Removal of damaged organelles. A double membrane forms an autophagosome, which fuses with a lysosome.

Lysosomes and Degradation

• Lysosomes: The primary site of intracellular digestion. Contain acid hydrolases (nucleases, proteases, glycosidases, lipases, etc.).
• Internal Environment: Maintain an acidic $\text{pH} \approx 5.0$ using an $H^+$ pump (ATP-driven). The cytosol has a $\text{pH} \approx 7.2$.
• Sorting Signal: Mannose-6-phosphate is added in the Golgi to direct enzymes to lysosomes.
• Lysosomal Storage Diseases: Examples include Gaucher disease.

Questions & Discussion

• Question 15.1: Drug blocks Ran's ability to exchange GDP for GTP. Result: (c) Nuclear transport receptors would be unable to release their cargo in the nucleus (since Ran-GTP is required for release).
• Question 15.2: Removal of N-terminal signal from mitochondrial protein. Result: (D) The protein will fail to be targeted efficiently to the mitochondrion.
• Question 15.3: Soluble protein "Fuzzy" in ER lumen. Result: (A) Fuzzy contains an N-terminal ER signal sequence that is recognized by SRP.
• Question 15.4: Protein with N-terminal ER signal (cleaved) and internal stop-transfer sequence. Result: (B) The C-terminus is in the cytoplasm, and the N-terminus is in the ER lumen.
• Question 15.5: Cell forms coated pits but no vesicle pinching/coat removal. Lacking protein: (c) dynamin.
• Question 15.6: FALSE statement about ER quality control. Answer: (E) The unfolded protein response completely shuts down all protein synthesis in the cell (it slows it down, but does not completely shut down all synthesis forever).
• Question 15.7: Process balancing the fluid intake of pinocytosis. Answer: (c) exocytosis.
• Study Question (Krt1): Vesicle orientation shows N-terminus in lumen. Upon fusion: (c) The N-terminus of Krt1 will be in the extracellular space.
• Study Question (Endosome Cargo): Factor causing receptor to release cargo: (b) acidic pH.
• Study Question (Lysosome Routing): Signal diverting proteins to lysosome: (d) a mannose-6-phosphate sugar added in the Golgi.
• Study Question (Secretion): True statement: (a) The membrane of a secretory vesicle fuses with the plasma membrane when the vesicle discharges its contents to the cell’s exterior.