Lecture 8
Protein Sorting: The Golgi Apparatus
Overview of the Secretory Pathway
The secretory pathway represents the route proteins take from synthesis to secretion. It comprises sequential steps:
Translation from mRNA occurs in the ribosomes located in the cytoplasm.
Newly synthesized proteins enter the endoplasmic reticulum (ER) lumen.
Proteins exit the ER and are transported to the Golgi apparatus in vesicles.
Proteins transit through the different compartments of the Golgi apparatus.
Proteins leave the Golgi in vesicles.
Secretory vesicles fuse with the cell membrane.
The proteins are released into the extracellular space.
Part 1: Vesicular Transport
Vesicular Transport Definition: Transport vesicles are membrane-bound carriers that bud off from one compartment and fuse with another, carrying material as cargo.
Pathway Types
There are two main pathways concerning intracellular transport:
Secretory Pathway: Leads outward from the ER to the Golgi apparatus and ultimately to the plasma membrane.
Endocytic Pathway: Moves inward, from the plasma membrane to the inside of the cell.
Coated Vesicles
Most transport vesicles originate from specialized, coated regions of membranes where they bud off as coated vesicles.
This coating provides a distinctive structure of proteins around the vesicle.
Types of Coated Vesicles: There are four well-characterized types:
Clathrin-coated vesicles
COPI-coated vesicles
COPII-coated vesicles
Retromer-coated vesicles
Each type is employed for different transport steps within the cell.
Vesicle Coats
Coats consist of geometrical structures that assemble around vesicle cages.
Typical size of a coated vesicle is about 50 nm.
Example of a coat protein: Clathrin.
Role of Rab Proteins
Rab Proteins: These proteins guide transport vesicles to their target membranes.
Transport vesicles possess surface markers that identify them, which are recognized by complementary receptors on target membranes.
SNARE Proteins and Membrane Fusion
SNARE proteins facilitate the fusion of membranes by closely positioning them.
Key stages include:
Docking Phase: Membranes are brought into proximity.
Fusion Phase: Membranes merge, facilitating content transfer.
Diverse protein configurations contribute to this process, including the trans-SNARE complex.
COPII-Coated Transport Vesicles
COPII-coated vesicles transport proteins out of the ER. They form at specific sites called ER exit sites (ERES).
Key components include:
Outer COPII coat proteins
Sar1-GTP as a small GTPase
Adaptor proteins
Resident ER proteins and cargo receptors ensuring specificity for cargo destined for transport.
KDEL Sequence: An important peptide that signals ER-resident proteins to return to the ER.
Sequence consists of Lysine (L), Aspartic Acid (D), Glutamic Acid (E), Leucine (L).
Part 2: The Golgi Apparatus
Discovery: Investigated by Camillo Golgi, who received the Nobel Prize in 1906 for his work on the apparatus and the use of osmium tetroxide staining methods.
Structure of the Golgi Apparatus:
Composed of flattened, membrane-enclosed compartments called cisternae.
Cisternae structure includes:
Cis Golgi Network (CGN): The entry face of the Golgi.
Medial Cisternae: Middle compartments for processing.
Trans Golgi Network (TGN): The exit face of the Golgi.
Transport vesicles and maturation processes occur here, marked by a typical size of around 1 μm in diameter.
Glycosylation and Phosphorylation
The Golgi is crucial for post-translational modifications like glycosylation and phosphorylation, which serve as "destination codes" for proteins.
Sugar coats function as tags for determining the final destination of proteins.
Modifications occur in specific compartments, illustrating the compartmentalization of processes during transport.
Mechanisms of Transport Through the Golgi
Two primary mechanisms are in effect during transport:
Vesicular Transport: Vesicles transport molecules between the cisternae.
Cisternal Maturation: Cisternae mature from the cis to the trans side in conjunction with cargo molecules.
Evidence of Models of Transport
Original Evidence: Early studies based on fixed samples supported the vesicular model and maturation model, indicating that vesicles bud and fuse at various stages.
Evidence demonstrated that COPI vesicles, typically between 60-90 nm, surround the Golgi and are involved in retrograde transport (back to the ER).
Proto Collagen (PC): This molecule, larger than 300 nm, was not found in vesicles but instead localized within expanded Golgi cisternae, providing insights into Golgi functionality.
Recent Imaging Techniques
Recent advancements like fluorescent live imaging techniques have allowed visualization of Golgi dynamics.
Fusion proteins can be created, such as those with Golgi proteins tagged with GFP. This enables real-time tracking of proteins through the Golgi, leading to the understanding of processes in various colors (indicating different markers/proteins).
Variability in Golgi Structure
In some organisms, such as the budding yeast Saccharomyces cerevisiae, the Golgi structure is dispersed, indicating variability in the organization of the Golgi apparatus across different species.
Analysis of Cisternae Dynamics
Fluorescence microscopy can also reveal the maturation dynamics of individual cisternae, showing differences in protein distribution between cis-Golgi (green) and trans-Golgi (red) proteins.
Retrograde Transport
COPI vesicles play a crucial role in retrograde transport, bringing ER proteins back from the Golgi to maintain cellular function and homeostasis.
Summary of Golgi Functions
Functions:
Molecules, particularly proteins, are sorted at the Golgi.
Glycosylation plays a key role as the main destination tag.
The Golgi has distinct parts including cis and trans networks (CisGN, TransGN).
Key Coats: COPI, COPII, and Clathrin facilitate various transport pathways.
The secretory pathway only accounts for a portion of cellular transport; additional mechanisms engage cytoplasmic and nuclear protein distribution, highlighting complexity within cell biology and cellular communication pathways.