Question

Cell Biology Tutorial: Vesicle Budding and Fusion

Answer 1

In introductory biology classes, you may be taught about cells forming vesicles to transport molecules into, out of, and within the cell. This tutorial will explore this concept further by explaining the molecular mechanisms in more detail.

Answer 2

First, we will discuss how the "vesicle coat" is formed, which describes the protein content of the vesicle surface; the vesicle coat determines the type of vesicle, which molecules are admitted into the vesicle, and the curvature of the vesicle.

Answer 3

v-SNARES are proteins that are directly involved in fusion with target membranes. After the coat is shed, v-SNARES join with t-SNARES (we call these SNARE-pairs). The joining of the SNARE pairs brings the vesicle membrane and the target membrane close together so fusion can occur.

Answer 4

There are three types of vesicles:

COPII: transport from ER to Golgi
COPI: cis-Golgi to ER; "retrograde" direction, meaning from the later portions of the golgi to the earlier
Clathrin-coated: trans-Golgi to endosomes; from plasma membrane

Answer 5

The curvature of vesicles requires polymerization of the coat proteins to occur.

Answer 6

A series of GTPase proteins control protein coat synthesis. Two examples are ARF (COPI and clathrin vessicles) and Sar1 (COPII). The steps, simplified are:

1. The ER membrane protein called Sec12 catalyzes the release of GDP from Sar1⋅GDP and the binding of GTP.
2. GTP binding induces a conformational change in Sar1 that exposes the amhpiphatic N-terminus, attaches the Sar1⋅GTP complex to the ER membrane
3. Sar1⋅GTP polymerizes COPII subunits, forming the vesicle coat
4. Once the vesicles are released, Sar1⋅GTP is hydrolyzed to Sar1⋅GDP, leading to the coat being disassembled

Answer 7

ARF follows a similar mechanism with some key differences: a myristate anchor in the N-terminus of ARF attaches ARF⋅GDP to the golgi membrane. The GTP to GDP exchange allows for the N-terminus to be inserted into the bilayer. The ARF⋅GTP complex is bound tightly to the membrane, and presumably follows a similar mechanism to steps 3 and 4

Answer 8

In mutated forms of Sar1 and ARF, the vesicle coats form but they cannot be disassembled, so they cannot disassemble and fuse with target membranes.

Answer 9

\({\bf{intra-cell~targeting}}\)

To ensure that the right cargo is moved to the right membrane, cargo proteins have sorting signals on the cystolic side. Proteins in the lumen have their own luminal sorting signals which bind to the luminal domain of the cargo.

Rab proteins direct targeting. Rab⋅GDP is attached by its isoprenoid anchor into the vesicle membrane. Then, the complex is converted to its GTP form, which can then bind to Rab effectors, which determine how the vesicle is "docked" on the target membrane.

As usual, the complex is hydrolyzed and released, allowing for the cycle to repeat itself.

Answer 10

\({\bf{SNARE-pair~disassembly}}\)

To recycle the components of the SNARE-pairs, ATP must be expended. NSF and alpha-SNAP proteins are involved; NSF hydrolyzes ATP and alpha-SNAP allows the NSF to attach to the SNARE complex.

Answer 11

\({\bf{This~is~the~end~of~my~tutorial;~I~hope~you~found~it~helpful.}}\)

\({\bf{If~you~have~any~*relevant*~comments~or~questions~I~will~attempt}}\)

\({\bf{to~address~them~to~the~best~of~my~ability.}}\)

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Answer 12

The source of this information is \({\scriptstyle{Molecular~Cell~Biology~Eigth~Edition}}\)
\({\scriptstyle{Lodish,~et.al.}}\)