Intracellular Compartments and Protein Transport

Intracellular Compartments and Protein Transport

Overview of Organelles and Their Functions

• Eukaryotic cells are highly organized with various membrane-enclosed organelles, each with specific functions.
• Key organelles include:
  • Endoplasmic Reticulum (ER)
  • Golgi apparatus
  • Lysosomes
  • Endosomes
  • Mitochondria
  • Peroxisomes
  • Nucleus
  • Cytosol
  • Plasma membrane
• Table 15-1 summarizes the main functions of these organelles:
  • Cytosol: Metabolic pathways, protein synthesis, cytoskeleton.
  • Nucleus: Contains the main genome; DNA and RNA synthesis.
  • Endoplasmic Reticulum (ER): Lipid synthesis; protein synthesis for distribution to many organelles and the plasma membrane.
  • Golgi Apparatus: Modifies, sorts, and packages proteins and lipids for secretion or delivery to other organelles.
  • Lysosomes: Intracellular degradation.
  • Endosomes: Sorting of endocytosed material.
  • Mitochondria: ATP synthesis by oxidative phosphorylation.
  • Chloroplasts (in plant cells): ATP synthesis and carbon fixation by photosynthesis.
  • Peroxisomes: Oxidative breakdown of toxic molecules.

Organelle Volumes and Numbers

• Table 15-2 provides the relative volumes and numbers of major membrane-enclosed organelles in a liver cell (hepatocyte):
  • Cytosol: 54% of cell volume, approximately 1 per cell.
  • Mitochondria: 22% of cell volume, approximately 1700 per cell.
  • Endoplasmic Reticulum: 12% of cell volume, approximately 1 per cell.
  • Nucleus: 6% of cell volume, approximately 1 per cell.
  • Golgi Apparatus: 3% of cell volume, approximately 1 per cell.
  • Peroxisomes: 1% of cell volume, approximately 400 per cell.
  • Lysosomes: 1% of cell volume, approximately 300 per cell.
  • Endosomes: 1% of cell volume, approximately 200 per cell.

Protein Import Mechanisms

• Proteins are imported into membrane-enclosed organelles via three main mechanisms.
• All mechanisms require energy.
• Mechanisms 1 and 3: Protein remains folded during transport.
• Mechanism 2: Protein usually needs to be unfolded during transport.

Signal Sequences

• Signal sequences are short amino acid sequences that direct proteins to specific cellular compartments.
• Table 15-3 shows some typical signal sequences:
  • Import into ER: +H3N-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-
  • Retention in lumen of ER: -Lys-Asp-Glu-Leu-COO–
  • Import into mitochondria: +H3N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu-
  • Import into nucleus: -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val-
  • Export from nucleus: -Met-Glu-Glu-Leu-Ser-Gln-Ala-Leu-Ala-Ser-Ser-Phe-
  • Import into peroxisomes: -Ser-Lys-Leu-
• Positively charged amino acids are shown in red, negatively charged in blue, and hydrophobic amino acids in green.
• +H3N indicates the N-terminus of a protein; COO– indicates the C-terminus.
• Relocated signal sequence: The relocation of a signal sequence to a cytosolic protein redirects it to the ER.

Nuclear Transport

• The nuclear envelope consists of the inner and outer nuclear membranes, the nuclear lamina, and nuclear pores.
• Nuclear pores are gateways for transport into and out of the nucleus.
• Nuclear pore complexes contain cytosolic fibrils and a nuclear basket.
• Prospective nuclear proteins (cargo) have a nuclear localization signal that is recognized by nuclear import receptors.
• Energy supplied by GTP hydrolysis drives nuclear transport.
• Ran-GAP (GTPase-activating protein) and Ran-GEF (guanine nucleotide exchange factor) regulate the GTP-bound state of Ran.
• Ran-GDP dissociates from the receptor in the cytosol, while Ran-GTP binds to the receptor in the nucleus.

Mitochondrial Protein Import

• Mitochondrial precursor proteins are unfolded during import.
• Import receptors in the outer mitochondrial membrane bind to the import signal sequence of precursor proteins.
• Protein translocators in the outer (TOM) and inner (TIM) membranes facilitate translocation into the matrix.
• The signal peptide is cleaved off in the matrix, resulting in a mature mitochondrial protein.

ER Protein Synthesis

• Ribosomes are either free in the cytosol or bound to the ER membrane.
• mRNA encoding a protein with no ER signal sequence remains free in the cytosol.
• mRNA encoding a protein with an ER signal sequence is targeted to the ER.
• The ER signal sequence and SRP (signal recognition particle) direct a ribosome to the ER membrane.
• The SRP is displaced and released for reuse after binding to the SRP receptor.

Protein Translocation into the ER Lumen

• A soluble protein crosses the ER membrane and enters the lumen through a protein translocator.
• The ER signal sequence is cleaved by signal peptidase.
• A single-pass transmembrane protein is retained in the lipid bilayer via a hydrophobic stop-transfer sequence.
• A double-pass transmembrane protein has an internal ER signal sequence, which acts as a start-transfer sequence, and a hydrophobic stop-transfer sequence.

Vesicular Transport

• Transport vesicles carry soluble proteins and membrane between compartments.
• Vesicle budding is driven by the assembly of a protein coat.
• Vesicle docking depends on tethers and SNAREs.
• Exocytosis (secretion) and endocytosis (uptake) are key processes in vesicular transport.

Clathrin-Coated Vesicles

• Clathrin molecules form basket-like cages that help shape membranes into vesicles.
• Clathrin-coated vesicles transport selected cargo molecules.
• Adaptins link clathrin to cargo receptors.
• Dynamin is involved in vesicle formation.
• Uncoating releases the clathrin coat.
• Table 15-4 summarizes some types of coated vesicles:
  • Clathrin-coated (clathrin + adaptin 1): From Golgi apparatus to lysosome (via endosomes).
  • Clathrin-coated (clathrin + adaptin 2): From plasma membrane to endosomes.
  • COPII-coated (COPII proteins): From ER to Golgi cisterna.
  • COPI-coated (COPI proteins): From Golgi cisterna to ER.

Vesicle Docking and Fusion

• Rab proteins, tethering proteins, and SNAREs help direct transport vesicles to their target membranes.
• V-SNAREs on vesicles and t-SNAREs on target membranes mediate docking and fusion.
• Following vesicle docking, SNARE proteins catalyze the fusion of the vesicle and target membranes.

Secretory Pathways

• Most proteins are covalently modified in the ER.

Protein Modification in the ER

• Many proteins are glycosylated on asparagines in the ER.
• Each oligosaccharide chain is transferred as an intact unit from dolichol to the asparagine, catalyzed by oligosaccharyl transferase.
• The enzyme scans polypeptides for the target sequence as they enter the ER lumen.

ER Quality Control and Size

• Exit from the ER is controlled to ensure protein quality.
• The size of the ER is controlled by the demand for protein folding.
• Accumulation of misfolded proteins in the ER lumen triggers an unfolded protein response (UPR).
• Misfolded proteins activate sensor proteins, leading to the activation of chaperone genes.

Golgi Apparatus

• Proteins are further modified and sorted in the Golgi apparatus.
• The Golgi apparatus consists of the cis Golgi network, cis cisterna, medial cisterna, trans cisterna, and trans Golgi network.
• Secretory proteins are released from the cell by exocytosis.

Exocytosis

• Newly synthesized soluble proteins and plasma membrane lipids undergo constitutive secretion (unregulated exocytosis).
• Secretory proteins undergo regulated secretion (regulated exocytosis) from secretory vesicles, triggered by extracellular signals.

Endocytic Pathways

• Specialized phagocytic cells ingest large particles (phagocytosis).
• Fluid and macromolecules are taken up by pinocytosis.
• Receptor-mediated endocytosis provides a specific route into animal cells.
• Endocytosed macromolecules are sorted in endosomes.
• Lysosomes are the principal sites of intracellular digestion.
• LDL enters cells via receptor-mediated endocytosis.

Lysosomes

• A lysosome contains a large variety of hydrolytic enzymes, which are only active under acidic conditions.
• The lumen of the lysosome is maintained at an acidic pH by an ATP-driven H^+ pump.

Degradation Pathways

• Phagocytosis, endocytosis, and autophagy deliver materials to lysosomes for degradation.
• Phagosomes, early endosomes, late endosomes, and autophagosomes are involved in these pathways.
• Hydrolytic enzymes in lysosomes break down the materials.