Unit 03 pt1

Unit 03 Overview

• Unit Title: Intracellular Compartments

• Course: BIOL 331 – Molecular Cell Biology

• Textbook: MBoC (7th Ed): Chapter 12 pages: 683 – 745.

• Sources: Molecular Biology of the Cell, 6th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2022.

Outline of Topics

• Membrane-enclosed compartments

• Subcellular Compartments

• Different Modes of Protein Sorting

• Sorting Signals and Examples

• Characterizing Signal Sequences Experimentally

• Subcellular Fractionation

Membrane-Enclosed Compartments

• Organelle membranes significantly contribute to the total cellular membrane content in mass and area.

Sub-Cellular Compartments: Topological Equivalence

• Certain organelles may have evolutionary origins from:

  • Membrane invaginations

  • Endosymbionts (e.g., mitochondria and chloroplasts)

• Categories of Compartments:

  1. Nucleus and Cytosol

  2. Endoplasmic Reticulum, Golgi Apparatus, Endosomes, Lysosomes, Transport Vesicles, Peroxisomes

  3. Mitochondria

  4. Plastids

Different Modes of Protein Sorting

• Starting Point: All proteins are synthesized in the cytosol, except those few by mitochondrial or chloroplast ribosomes.

• Protein Destinations Include:

  • Nucleus

  • Mitochondria

  • Plasma Membrane

  • Secreted from the cell

• Sorting Signals:

  • Specific amino acid sequences that determine protein destination.

  • Must have a corresponding receptor for signal recognition.

  • Proteins without sorting signals, like actin, remain in the cytosol.

  • Organelles contain necessary information and cannot be constructed de novo.

Modes of Protein Transport

• Gated Transport:

  • Nuclear pore complexes serve as gates for specific macromolecule transport.

  • Small molecules can diffuse freely.

• Transmembrane Transport:

  • Uses protein translocators to transport specific proteins across membranes.

  • Typically involves proteins being unfolded to pass through.

• Vesicular Transport:

  • Involves transport vesicles budding from one compartment to fuse with another.

  • Transfers soluble proteins, maintains membrane orientation for membrane proteins.

• Engulfment:

  • A double membrane surrounds and encloses material for a new compartment.

Sorting Signals

• Definition: Specific amino acid sequences (15-60 residues) that guide proteins to organelles.

• Types of Sorting Signals:

  • Signal Sequences: Typically at N-terminus, cleaved after delivery.

  • Internal Sequences: May remain part of the mature protein or present at the C-terminal.

  • Signal Patches: 3-D conformations, not necessarily based on contiguous sequences.

Examples of Signal Sequences

• Key Signals to Remember:

  • Basic amino acid clusters for nuclear import

  • KDEL for proteins returning to ER

• Role of Sorting Receptors:

  • Guide sorting signals to destinations and recycle after unloading cargo.

  • Most receptors recognize a class of proteins rather than being highly specific.

Amino Acids Overview

• Single Letter Code Highlights:

  • Cys (C), His (H), Ile (I), Met (M), Ser (S), Val (V)

  • Commonly occurring: Ala (A), Gly (G), Leu (L), Pro (P), Thr (T)

  • Phonetics: Phe (F), Arg (R)

  • W for Trp due to structure

• General representation: X = any amino acid

Characterizing Signal Sequences Experimentally

• Approach:

  • Use recombinant DNA to fuse a signal sequence with a cytosolic protein sequence, creating a fusion protein.

  • Introduce into cells (through transfection, etc.) for expression.

  • Investigate localization via immunostaining, cell fractionation, or fluorescence microscopy.

  • Test changes in amino acid sequence to uncover structural signaling function.

Subcellular Fractionation

• Cells can be disrupted through various methods (e.g., osmotic shock, sonication).

• Resulting homogenate can be separated via differential centrifugation, isolating components by size and density.

• Techniques Utilized:

  • Western blotting with antibodies or tags to identify proteins in fractions.

  • Measure enzyme activities in separated fractions, providing insight into various cellular functions.