Biol 5210: Protein Synthesis and Transport Notes

Endoplasmic Reticulum (ER) Overview

  • Functions of the ER:
    • Protein translocation and folding
    • Protein glycosylation
    • Protein quality control
    • Lipid synthesis and homeostasis
    • Calcium ion storage and release
  • Composition:
    • Represents 30-50% of total cellular proteins depending on cell type and stage.

Protein Translocation

  • Signal Recognition Particle (SRP):
    • Targets proteins with a hydrophobic N-terminal signal sequence for translocation to the ER membrane.
  • Types of Proteins Translocated:
    • Secretory proteins (e.g., insulin, antibodies)
    • Integral membrane proteins (e.g., GPCRs)
    • Lysosomal proteins
    • Resident ER and Golgi proteins

Membrane Protein Topology

  • Type I Membrane Proteins:
    • Contain N-terminal signal sequence for ER localization.
  • Type II Membrane Proteins:
    1. Translated in cytosol until an internal signal anchor sequence is recognized.
    2. SRP directs the ribosome to the ER.
    3. Translation/translocation continues into the ER.
  • Hydropathy Profiles:
    • Positive values indicate hydrophobic regions.

Isolation of ER Fragments

  • Sucrose Gradient Centrifugation:
    • Technique to isolate ER proteins by density.
    • ER vesicles generated through cell homogenization are referred to as microsomes.

Vesicle Budding and Fusion

  • COPII and COPI Key Roles:
    • Pack soluble cargo into vesicles with the help of membrane receptors.
    • COPII does not require dynamin for vesicle pinching.
  • G Protein Regulation:
    • A regulatory G protein switch determines vesicle affinity and disassembly.

COPII Coat Assembly

  • Key Players:
    • Small GTPase Sar1 regulates COPII assembly:
    • Inactive Sar1 is soluble, while its active form interacts with the ER membrane, inducing curvature.
    • Other proteins like Sec23 and Sec24 are recruited for further curvature and functionalization of vesicle coats.
  • Coat Disassembly:
    • GTP hydrolysis of Sar1 leads to uncoating before vesicle fusion with target membranes.

Membrane Fusion Mechanism

  • SNARE Proteins:
    • Critical for targeting and fusion; exist as v-SNAREs (vesicle) and t-SNAREs (target).
    • Example: Fusion of secretory vesicles with the plasma membrane involves distinct SNARE pairings.

Golgi Complex Functions

  • Protein Sorting and Glycosylation:
    • Involves KDEL receptors for ER resident proteins and regulates their transport back from the Golgi to ER.
  • Models of Transport:
    • Vesicular Transport Model: forward (COPII) and retrograde (COPI) transport of vesicles.
    • Cisternal Maturation Model: golgi cisternae mature and progress through the stack while carrying cargo.

Protein Glycosylation

  • Importance of Glycosylation:
    • Changes protein surface properties, aids in cell-cell contact and immune recognition.
    • Enhances protein solubility and stability: protects from proteases and serves as a sorting signal.
  • N-linked Glycosylation:
    • The predominant form, occurs co-translationally, with sugars modified in the ER or Golgi.

Key Learning Objectives

  1. Understand ER functions and its membrane composition.
  2. Distinguish properties of protein types and their synthesis machinery.
  3. Interpret protein hydropathy plots and describe techniques to assess membrane orientation.
  4. Explain steps in COPII coat assembly and the role of GTPases.
  5. Identify major SNARE proteins involved in vesicle fusion.
  6. Comprehend the Golgi complex functions and models of protein processing.
  7. Recognize the implications of protein glycosylation in cellular function and structure.