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Chapter 13

Chapter 13.docx


Department
Biology
Course Code
BIOL331
Professor
Moira Glerum
Chapter
13

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Chapter 13: Intracellular Vesicular Transport
The overall organization of membranes in a eukaryotic cell is better thought as a system of
membranes.
Cells need to eat, communicate and respond to changes in its environment.
o They do most of these things through the membrane system
Adjusting the composition of the plasma membrane
Use of an internal membrane system to add/remove cell-surface proteins
embedded in the membrane
Exocytosis- biosynthetic-secretory pathway delivers newly synthesized proteins, carbohydrates,
and lipids to either the plasma membrane or the extracellular space.
Endocytosis
o Cells remove/excise plasma membrane components and deliver them to internal
compartments called endosomes, from where they can be recycled to the same or
different regions of the plasma membrane or can be delivered to lysosomes for
degradation.
o Capture nutrients, such as vitamins, lipids, cholesterol, and iron; taken up together with
the macromolecules to which they bind and then released in endosomes or lysosomes and
transported into the cytosol, where they are used in various biosynthetic processes.
Interior space, or lumen, of each membrane-enclosed compartment along the biosynthetic-
secretory and endocytic pathways is topologically equivalent to the lumen of most other
membrane-enclosed compartments and to the cell exterior.
o Proteins travelled in this space without having to cross a membrane, being passed from
one compartment to another by transport vesicles.
Transport Vesicles: membrane enclosed containers that can be small spherical
vesicles or large irregular vesicles or tubules formed from the donor compartment.
Transport vesicles continually bud off from one membrane and fuse with another, carrying
membrane components and soluble molecules,
which are referred to as cargo
Biosynthetic-secretory pathway:
o Leads outward from the ER toward the
Golgi apparatus and cell surface with a side
route leading to lysosomes
Endocytic Pathway
o Leads inward from the plasma membrane
Retrieval Pathway
o Balances the flow of membrane between
compartments in the opposite direction
o Brings membrane and selected proteins
back to the compartments of origin.
Transport vesicles are highly selective (must take
up only the appropriate molecules and must fuse
only with the appropriate target membrane)
The Molecular Mechanisms of Membrane
Transport and the Maintenance of Compartmental
Diversity

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There are about ten distinct membrane compartments that use vesicular transport (to mediate a
continuous exchange of components)
Compartment- the cytosolic surface of the compartment membrane carries molecular markers
that serve as guidance cues for incoming vesicles. Many membrane markers are found on more
than one compartment so it is the distinct combination of molecular markers that generates
specificity.
How does each compartment then acquire its molecular markers?
o The answer relates to how patches of membrane, enriched or depleted in specific
components, bud off from one compartment and transfer to another.
How do cells segregate components into membrane domains?
o Proteins are assembled into domains following the assembly of a special protein coat on the
cytosolic surface of the membrane.
Cell-Free Systems for Studying Vesicular Transport
First achieved for the Golgi Stack
o Golgi stacks are isolated from cells and incubated with cytosol and with ATP (energy)
Transport Vesicles bud from their rims and appear to transport proteins between
cisternae.
Follow the process of vesicular transport by monitoring the progressive processing of the
oligosaccharides on a glycoprotein as it moves from one Golgi component to the next.
Two distinct populations of Golgi stacks are incubated together
1. “Donor” population is isolated from mutant cells that lack the enzyme N-
acetylglucosamine (GlcNAc) transferase I and that have been infected with a virus
- Because of the mutation, the major viral glycoprotein fails to be modified with
GlcNAc in Golgi apparatus of the mutant cells.
2. “Acceptor” Golgi stacks are isolated from uninfected wild-type cells and thus contain a
good copy of GIcNAc transferase I, but lack the viral glycoprotein
In the mixture of Golgi stacks, the viral glycoprotein acquires GIcNAc, indicating that it must have
been transported between the Golgi stacks presumably by vesicles that bud from the cis
compartment of the donor Golgi and fuse with the medial component of the acceptor Golgi or by
homotypic fusion between the cisternae of two Golgi stacks.
Transport-dependent glycosylation is monitored by measuring the transfer of 3H-GlcNAc from
UDP-3H-GlcNAc to the viral glycoprotein.
o Transport occurs only when ATP and cytosol are added
By fractionating the cytosol, a number of specific proteins have been identified that
are required for the budding and fusion of transport vesicles.
Genetic Approaches for Studying Vesicular Transport
Studies of mutant yeast cells defective for secretion have identified more than 25 genes that
are involved in the secretory pathway.
Many of the mutant genes encode temperature-sensitive proteins.
o Functions normally at 25°C, but when the mutant cells are shifted to an elevated
temperature, such as 35°C, they fail to transport proteins from the ER to the Golgi,
others from one Golgi cisterna to another, and still others from the Golgi apparatus to
the vacuole (the yeast lysosome) or to the plasma membrane.
Multicopy suppression used to identify genes that encode other proteins that interact with it.
At high temperatures, a temperature-sensitive mutant protein often has too low an affinity
for its normal interaction partners.
o If interacting proteins are produced at much higher concentrations, sufficient binding
occurs to cure the defect.
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To create an experimental paradigm in which such high concentrations of ligand are present,
mutant yeast cells (with a temperature-sensitive mutation in a gene involved in vesicular
transport) are transfected with a yeast plasmid vector to which random normal yeast
genomic DNA fragments have been cloned.
o Because these plasmids are maintained in cells at high copy number, any cells that
happen to carry plasmids with intact genes will overproduce the normal gene product,
allowing rare cells to survive at the high temperature
Relevant DNA fragments, which presumably encode proteins that interact with the original
mutant protein, can then be isolated from the surviving cell clones.
GFP Fusion Proteins have revolutionized the Study of Intracellular Transport
Green fluorescent protein (GFP) is attached by genetic engineering techniques to the protein
of interest
o When a cDNA encoding such a fusion protein is expressed in a cell, the protein is
readily visible in a fluorescent microscope.
GFP fusion proteins are widely used to study the location and movement of proteins in cells.
o GFP fused to proteins that shuttle in and out of the nucleus. (Ex. Facilitates studies of
nuclear transport and its regulation)
o GFP fused to mitochondrial or Golgi proteins is used to study the behaviour of these
organelles
o GFP fused to plasma membrane proteins allows measurement of the kinetics of their
movement from the ER through the secretory pathway.
Study of GFP fusion proteins is often combined with FRAP and FLIP techniques, in which the
GFP in selected regions of the cell is bleached by strong laser light.
o Rate of diffusion of unbleached GFP fusion proteins into that area can then be
determined to provide measurements of the protein’s diffusion or transport in the cell.
Example: determined that many Golgi enzymes recycle, between the Golgi
apparatus and the ER.
(VSV in ER) Cultured cells express a GFP fusion protein consisting of GFP attached to a viral coat
protein (Vesicular Stomatitits)
Viral protein is an integral membrane protein that normally moves through the
secretory pathway from the ER to the cell surface, where the virus would be
assembled if cells also expressed the other viral components
Viral proteins contain mutation that allows export from the ER only at a low
temperature.
o At high temperatures, the fusion protein labels the ER
(VSV leaves ER) As temperature is lowered, the GFP fusion protein rapidly accumulates at ER
exit sites
(VSV in Golgi) Fusion protein then moves to the Golgi apparatus
(VSV in Plasma Membrane) Fusion protein is delivered to the plasma membrane where the
delivered protein diffuses into the plasma membrane
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