Lecture 10: SNAREs I

Aims

To consider how proteins are sorted in compartmentalised eukaryotic cells and to describe similarities with prokaryotic systems.

Learning Objectives

● To understand nuclear import and export

● To describe protein import into mitochondria, chloroplasts and bacteria

● To understand mechanisms of ER translocation

Physiological Significance of Membrane Fusion

Membrane fusion is a continuous, vital process within the human body required for numerous biological functions:

Synaptic Vesicle Fusion: Essential for communication between neurons and between neurons and muscle cells.
Secretory Granule Fusion: Occurs in both the endocrine and exocrine pancreas.
Serum Protein Secretion: Includes the release of albumin from hepatocytes and antibodies from plasma cells.
Mucus Secretion: Conducted by epithelial mucosal cells.
Intracellular Transport: The movement of proteins between various organelles within every cell.

Historical Overview and Research Methodologies

The ability to visualize secretory vesicles began with the development of the electron microscope in $1938$ , which allowed researchers to see membrane vesicles directly. The transition from observing microscopic anatomy to understanding the molecular machinery of vesicle fusion was achieved through three primary scientific approaches:

Biochemical Reconstitution: Purifying and assembling components in a cell-free system to reproduce a biological process.
Yeast Genetics: Identifying genes required for secretion by isolating mutants.
Cloning: Identifying and sequencing the specific proteins involved in the fusion machinery.

Biochemical Reconstitution: The Intra-Golgi Transport Assay

Jim Rothman utilized cell-free reconstitution to discover the proteins underlying intracellular transport. This approach was modeled after historical biochemistry techniques, such as the discovery of alcoholic fermentation in yeast extracts at the end of the $19^{th}$ century. Despite skepticism that destroying spatial relationships via homogenization would halt transport, Rothman successfully isolated the protein machinery.

The Experimental Design (1984):

Donor Golgi Fraction: Purified from a $15B$ mutant cell line infected with Vesicular Stomatitis Virus (VSV). This mutant is missing the key glycosylation enzyme $GlcNAc$ Transferase ( $GlcNAc \text{ Tr'ase}$ ).
Reporter Molecule: The Vesicular Stomatitis Virus Glycoprotein G ( $VSV-G$ protein) acts as a spike molecule, trafficking from the ER to the Plasma Membrane (PM).
Acceptor Golgi Fraction: Obtained from uninfected wild-type cells that contain the $GlcNAc$ Transferase enzyme.
The Assay: Donor and acceptor Golgi are mixed with ATP, cytosol, and a radioactive sugar (UDP- $GlcNAc$ ). Transport is measured by the incorporation of the radioactive sugar into the $VSV-G$ protein once it moves from the donor to the acceptor compartment where the enzyme resides.

Discovery and Identification of NSF and SNAPs

NSF (N-ethylmaleimide Sensitive Factor, 1988): It was discovered that the alkylating reagent N-ethylmaleimide (NEM) inhibits the fusion reaction. The target of this inhibition was purified and named NSF. NSF is an ATPase.
SNAP (Soluble NSF Attachment Protein, 1990): When membranes are salt-washed, NSF can no longer bind to them. The factor required for NSF binding was purified and named SNAP.
The 20S Fusion Particle: NSF, SNAP (specifically $\alpha$ and $\gamma$ isoforms), and a previously unidentified SNAP Receptor ( $SNARE$ ) form a large complex called the $20S$ complex. NSF cycles on and off membranes in an ATP-dependent manner, requiring ATP hydrolysis.

Yeast Genetics and the Isolation of SEC Mutants

Randy Schekman (1979-1980) used yeast genetics to identify the machinery of secretion. He isolated $sec$ (secretory) mutants that failed to transport proteins. It was later discovered that these genetic findings converged with biochemical findings:

SEC1: A SNARE-binding protein.
SEC17: Encodes $\alpha-SNAP$ .
SEC18: Encodes NSF.

Cloning and Synaptic Protein Identification

In $1988$ , Richard Scheller and Reg Kelly used antibodies raised against purified synaptic vesicles from the Pacific electric ray to expression clone key synaptic proteins:

VAMP (Vesicle-Associated Membrane Protein): Also known as Synaptobrevin.
Syntaxin: A protein located on the target membrane.

In $1992$ , Cesare Montecucco demonstrated that clostridial neurotoxins (Tetanus and Botulinum B) specifically cleave VAMP, providing evidence for its critical role in fusion.

The SNARE Hypothesis

In $1993$ , Jim Rothman purified a large complex containing Syntaxin, VAMP, and SNAP-25 ( $Synaptosomal\text{ associated protein of 25 kD}$ ). This led to the SNARE hypothesis:

Unique Pairs: There are specific SNAREs for each transport step within the cell.
Specificity: SNAREs provide the specificity required for vesicle transport to the correct destination.
Sufficiency: SNAREs alone should be sufficient to drive the fusion of lipid bilayers.
Original (Incorrect) Proposal: It was initially proposed that NSF and ATP hydrolysis catalyzed the fusion step itself. This was later corrected; NSF actually catalyzes the disassembly and recycling of SNAREs after fusion.

SNARE Structure and Classification

Physical Features:

SNAREs are generally small proteins, ranging from $14$ to $40 \text{ kDa}$ .
They possess at least one coiled-coil or SNARE motif.
Most are C-terminally anchored to the membrane.

The Re-classification (R and Q SNAREs): SNAREs are divided based on a highly conserved salt bridge in the center of the complex (the "zero layer"):

R-SNAREs: Contain an Arginine ( $R$ ) residue at the center. Typically located on the vesicle (e.g., every VAMP is an R-SNARE).
Q-SNAREs: Contain a Glutamine ( $Q$ ) residue. Typically located on the target ( $T$ ) membrane.
- Qa: Syntaxin 1a ( $Q226$ ).
- Qb: SNAP-25-N ( $Q53$ ).
- Qc: SNAP-25-C ( $Q174$ ).
Conserved Ratio: Every functional SNARE complex maintains a ratio of $3Q:1R$ . Mutations in the $Q$ or $R$ residues within the coiled-coil domains inhibit SNARE activity.

Mechanisms of Membrane Fusion

Axel Brunger (1998) solved the crystal structure of the neuronal SNARE complex, revealing a parallel coiled-coil arrangement of Syntaxin, VAMP, and SNAP-25.

Zippering: SNAREs "zipper up" from the amino-termini toward the carboxy-termini. This zippering provides the necessary energy to overcome the energetically unfavorable barrier of bringing two negatively charged membranes together.
Trans-SNARE Complex: The complex formed when SNAREs from opposing membranes (vesicle and target) begin to link, before fusion occurs.
Cis-SNARE Complex: The complex formed after fusion, where all SNAREs are located on the same integrated membrane.
Recycling: NSF acts on the cis-SNARE complex after fusion to "unscrew" or disassemble the complex in an ATP-dependent manner, allowing SNAREs to be reused for future rounds of fusion.

Specificity and Functional Testing

Human Genome: Encodes approximately $38$ different SNAREs involved in various transport steps (e.g., $STX6$ , $STX10$ ).
Specificity: While SNAREs show some promiscuity in vitro, they predominantly interact with appropriate partners. Specificity is further bolstered by additional machinery like Rabs, coat proteins, and tethering factors.
Minimal Fusion Machinery: Using TIRF ( $Total\text{ Internal Reflection Fluorescence}$ ) microscopy, it was demonstrated that recombinant SNAREs are the minimal machinery required to drive the fusion of purified liposomes. While calcium is not required for the basic SNARE-driven fusion, it often acts as a regulator in physiological systems (e.g., through Synaptotagmin).

The 2013 Nobel Prize in Physiology or Medicine

The Nobel Prize was awarded jointly to James E. Rothman, Randy W. Schekman, and Thomas C. Sdhof "for their discoveries of machinery regulating vesicle traffic, a major transport system in our cells."

Questions & Discussion

Regulation: How is SNARE function regulated in the cell (e.g., inhibition or activation at specific times)?
Trafficking: How are different SNARE proteins accurately trafficked to their specific target membranes?