Molecular and Cellular Biology Overview

Endoplasmic Reticulum and Protein Synthesis

Overview of Key Topics

• Endoplasmic Reticulum (ER)

• Protein Synthesis and Cotranslational Import

• Protein Targeting

Introduction to Molecular and Cellular Biology

• Kinesin molecule transporting a vesicle: illustrates mechanisms of intracellular transport.

Apoptosis

• Definition: Apoptosis is an important, regulated process that eliminates redundant or damaged cells.
    - Aberrant apoptosis is associated with diseases:
      - Insufficient apoptosis can lead to cancer.
      - Excessive apoptosis can lead to neurodegenerative diseases.

• Caspases: These are proteases that activate proteins involved in dismantling the cell, playing a critical role in the apoptosis process.

Endomembrane System

• The endomembrane system mediates two key processes:
    - Exocytosis: The process by which materials are exported from the cell.
    - Endocytosis: The process by which materials are brought into the cell via vesicular transport.

• Visualization: Protein traffic patterns can be visualized using Green Fluorescent Protein (GFP).

Endoplasmic Reticulum (ER)

General Structure and Function

• The ER is continuous with the nuclear envelope and comprises two main types:
    - Rough Endoplasmic Reticulum (RER): Contains ribosomes on its cytoplasmic surface and is responsible for:
      - Synthesizing proteins destined for secretion and membrane insertion.
    - Smooth Endoplasmic Reticulum (SER): Lacks ribosomes and is involved in:
      - Lipid synthesis.
      - Production of steroid hormones (e.g., glucocorticoids, androgens, estrogens) in endocrine cells.
      - Detoxification of drugs and poisons, particularly in liver cells containing enzymes for modifying foreign compounds.
      - Sequestration (storage) of Ca²⁺ ions.

Functions of Smooth ER (SER)

1. Lipid Synthesis: Production of various lipids.

2. Hormone Production: Including steroid hormones.

3. Detoxification: Enzymes that modify harmful substances in liver cells.

4. Calcium Storage: Maintaining low calcium concentrations in cytosol to prevent precipitation of phosphates.

Calcium Ion Regulation

• Cells utilize considerable energy to regulate changes in calcium ion ($Ca^{2+}$) concentration.

• Reasons for $Ca^{2+}$ exclusion from cytosol include:
    - Poor binding with water leading to insoluble phosphate precipitation.
    - Utilization of Ca²⁺ binding proteins and pumps to manage intracellular calcium levels.
    - Specific organelles function as calcium 'sinks' or 'pools' for storage and regulation.

Functions of Rough ER (RER)

1. Membrane Phospholipid Synthesis: Key for forming cell membranes.

2. Glycosylation of Proteins:
      - Involves addition of carbohydrate chains to specific proteins, crucial for their function and stability.

3. Protein Folding:
      - Quality control mechanisms assist in proper protein folding, involving molecular chaperones.

4. Protein Synthesis, Modification, and Transport:
      - Proteins are synthesized and properly modified to be directed to various cellular compartments (ER, Golgi, plasma membrane).

Overview of Protein Synthesis

• Transcription:
    - RNA polymerase reads a DNA sequence to produce a complementary, antiparallel strand of RNA.
    - Eukaryotic cells modify the primary RNA transcript in the nucleus, which results in mature mRNA and noncoding RNA (e.g., tRNA and rRNA).

• RNA Processing:
    - Introns are excised, exons spliced, and the RNA is modified with a 5' cap and poly(A) tail.
    - mRNA exits the nucleus through nuclear pores into the cytoplasm.

Translation Process

Steps of Translation:

1. Initiation:
      - A ribosome binds to mRNA and scans for an AUG start codon.

2. Elongation:
      - tRNA molecules transport amino acids to the ribosome, matching anticodons with codons on mRNA.
      - Peptide bonds form between amino acids, extending the polypeptide chain.

3. Termination:
      - Translation terminates upon reaching a stop codon (UAA, UAG, UGA).

Ribosome Structure

• The ribosome consists of two subunits (60S and 40S) which house different sites during protein synthesis:
    - P-site (Peptidyl): Where the growing peptide chain is held.
    - A-site (Aminoacyl): Where the new tRNA carrying the next amino acid arrives.
    - E-site (Exit): Where the uncharged tRNA exits.

Types of Ribosomes

• Free Ribosomes: Synthesize cytosolic proteins, peripheral membrane proteins, and proteins targeted to organelles like the nucleus and mitochondria.

• ER Bound Ribosomes: Synthesize proteins destined for secretion or integration in membranes of the endomembrane system.

Cotranslational Protein Import

• Involves targeting the ribosome-mRNA-polypeptide complex to the ER:
    1. Signal Sequence: An N-terminal sequence directs the nascent polypeptide to the ER.
    2. Signal Recognition Particle (SRP): Binds the signal sequence and halts translation.
    3. Translation Resume: Once the ribosome is bound to the ER, translation resumes, and the polypeptide is translocated into the ER lumen through the translocon.
    4. Cleavage and Folding: After entering the ER, the signal peptide is cleaved, and chaperones assist in folding the protein.

Protein Targeting Beyond the ER

• Proteins synthesized on free ribosomes that target mitochondria or chloroplasts utilize different mechanisms (e.g., TOM complex).

• Post-synthesis:
    1. Proteins can be retained in the ER or transported to the Golgi complex for further modification.
    2. These can eventually be delivered outside the cell for secretion, such as steroid hormones or mucins.

Study Questions

• Synthesis of Proteins in Mitochondria: Identify the strategies for targeting.

• Cytochrome c: Discuss its cytoplasmic presence and implications.

• Integral Membrane Protein: Understanding the surface outcomes when tagged with GFP.

• Overall Initiation: Where does all protein synthesis begin?

Suggested Readings

• Morris Chapter 5, Section 5.4

• Lodish Chapter 13, Figure 13-1 and Section 13-1

• Chapter 14, Introduction (pages 631-634)