Cell Systems II: The Endomembrane System

7.5 Cell Systems II: The Endomembrane System Manufactures, Ships, and Recycles Cargo

Introduction to Cargo Movement in Cells

  • Cargo movement occurs in a regulated and energy-demanding manner not only at the nuclear membrane but also within various cellular compartments.

  • Key organelles involved include peroxisomes, mitochondria, and chloroplasts, which primarily import proteins made by cytosolic free ribosomes.

Importance of Protein Sorting and Transport

  • Proteins synthesized in the cytosol must be sorted and shipped to specific organelles:

    • Acid hydrolases are transported to lysosomes.

    • Catalase is transported to peroxisomes.

  • Each protein has a specific “zip code” for targeting and a delivery system for transport.

Overview of the Endomembrane System

  • The endomembrane system is crucial for protein synthesis, processing, and transport.

  • Proteins synthesized on the rough endoplasmic reticulum (ER) move to the Golgi apparatus for further processing before being dispatched to their final destinations.

The Secretory Pathway Hypothesis

  • Inspired by observations of cells that secrete substances:

    • Cells rich in rough ER and Golgi are likely involved in a “secretory pathway.”

    • The pathway initiates at the rough ER and culminates in the secretion of products from the cell.

Tracking Protein Movement: The Pulse–Chase Assay

Overview of Pulse–Chase Experiment

  • Conducted by George Palade and colleagues, this method tracked the movement of proteins over time.

  • Comprises two critical steps:

    1. Pulse: Cells are exposed briefly to a high concentration of modified amino acids, effectively labeling synthesized proteins (e.g., using radioactively labeled leucine).

    2. Chase: The modified amino acid is washed away and replaced with a non-labeled version, halting further labeling and allowing tracking of previously marked proteins.

  • Purpose: To observe the behavior of a specific cohort of proteins over time.

Results and Observations

  • Tracking primarily focused on pancreatic cells (known for secreting digestive enzymes):

    • Experimental timing involves a 3-minute pulse and a lengthy chase period.

    • The results indicated distinct patterns of protein trafficking through various organelles, observed using electron microscopy and autoradiography.

Observations from the Pulse–Chase Experiment

  • 0 Minutes: Labeled proteins reside mostly in the rough ER.

  • 37 Minutes: Labeled proteins are found in the Golgi apparatus and secretory vesicles.

  • 117 Minutes: Most labeled proteins exit the Golgi apparatus, either packaged into secretory vesicles or secreted from the cell.

Endomembrane Pathway Steps

Entry into the Rough ER

  • Protein synthesis starts in free ribosomes in the cytosol.

  • The signal hypothesis suggests that proteins have an ER signal sequence that guides them to the ER.

  • The sequence usually consists of the first 20 amino acids and is cleaved upon protein synthesis completion.

Steps for Protein Entry into the ER:

  1. Protein synthesis begins in the cytosol with the ribosome synthesizing the ER signal sequence.

  2. The signal sequence binds to a signal recognition particle (SRP), pausing protein synthesis.

  3. The SRP guides the ribosome to the ER membrane where it attaches to the SRP receptor.

  4. Upon connection, protein synthesis resumes through the translocon channel.

  5. The growing protein is fed into the ER lumen, and the signal sequence is removed.

  • Integral Membrane Proteins: Translocon’s gates aid in inserting nonpolar amino acids into the lipid bilayer, leaving integral membrane proteins attached.

Folding and Modification of Proteins in the ER

  • Proteins achieve their functional conformation with assistance from chaperone proteins.

  • Carbohydrate side chains are added during folding, a process termed glycosylation, leading to the formation of glycoproteins.

Movement Between the ER and Golgi Apparatus

  • Studies indicated that proteins move as cargo in vesicles which bud off from the ER to the Golgi apparatus.

  • Researchers used differential centrifugation to isolate and study these vesicles.

Golgi Anatomy and Function

  • The Golgi apparatus consists of a stack of flattened compartments called cisternae. Cargo enters on one side and exits on the opposite side.

  • Dynamic Composition: Cisternae are constantly formed and broken down, allowing stepwise modifications to cargo as it matures.

Sorting and Destination of Proteins

  • Cargo Sorting: Proteins that transit through the Golgi must be sorted and dispatched appropriately:

    • Hydrolytic enzymes bound for lysosomes receive a phosphate group attached to specific sugars, forming mannose-6-phosphate (the “zip code”).

    • This tag is essential for targeting proteins to the lysosomes.

  • Exocytosis is the process whereby vesicles fuse with the plasma membrane, releasing contents outside the cell, exemplified in pancreatic cells releasing digestive enzymes.