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 Vesicular traffic

Cell Biology: Lecture 5

 Vesicular transport

Each cell communicates with the extra cellular environment by transporting materials
form one place to another by using membrane bound containers called vesicles.

Eucaryotic cells have evolved an elaborate internal membrane system that allows them to
take up macromolecules by the process of endocytosis and deliver them to digestive
enzymes stored in lysosomes inside the cell

The biosynthetic-secretory pathway allows the cell to modify the molecules it produces
in a series of steps, store them until needed, and then deliver them to the exterior
through a specific cell-surface domain by a process called exocytosis
 Vesicles continually bud off from one membrane and fuse with another
Biosynthetic-secretory pathway:
outward from the ER toward the
Golgi apparatus and cell surface,
with a side route leading to
lysosomes
Endocytic pathway: leads inward
from the plasma membrane
Retrieval pathway: for the
backflow of selected components

Each transport vesicle that buds from a compartment must be selective. It must take up only
the appropriate proteins and must fuse only with the appropriate target membrane
Experiment to show protein transport
Experiment to show protein transport from ER to Golgi
Elucidation of different steps of protein transport through analysis of sec mutants
Experiment to show protein transport from Golgi to Golgi
 The Molecular Mechanisms of Transport

The sorting process depends on the assembly of a special protein coat on the cytosolic
face of the donor membrane

They bud off as coated vesicles that have a distinctive cage of proteins covering their
cytosolic surface. Before the vesicle fuses with a target membrane, the coat is discarded, as
is required to allow the two cytosolic membrane surfaces to interact directly and fuse
There are three types of coated proteins: clathrin-coated, COPI-coated, and COPII-
coated vesicles

Function of coat: selects the appropriate molecule, give shape of the vesicle
 Each coated vesicle is used for different transport steps in the cell

Clathrin-coated vesicles: mediate transport from the Golgi apparatus and from the
plasma membrane
COPI- and COPII-coated vesicles: most commonly mediate transport from the ER and
the Golgi cisternae
 The Assembly of a Clathrin Coated Vesicles

Each clathrin subunit consists of three large and three small polypeptide chains
that together form a three-legged structure called a triskelion

A second major coat protein in clathrin-coated vesicles is a multisubunit complex

called adaptin, positioned between the clathrin cage and the membrane

Adaptin is required both to bind the clathrin coat to the membrane and to trap various
transmembrane proteins, including transmembrane receptors that capture soluble
cargo molecules inside the vesicleso-called cargo receptors
 Budding off of vesicles mediated by clathrin and adaptin

Clathrin-coated vesicles budding from different membranes use different

adaptins and thus package different receptors and cargo molecules
Phosphoinositides mark membrane domains

Distribution of converting enzymes and

hence the PIPs varies from organelle to
organelle and often within one region to
another of a continuous membrane and thus
define the specialized membrane domain
 Pinching off

Mediated by soluble cytoplasmic

protein dynamin which has
PI(4,5)P2-binding domain
 Additional control:GTP-binding protein controls coat assembly
Coat-recruitment GTPases - act as molecular switches that flip between an active state with
GTP bound and an inactive state with GDP bound

Sar 1 is a coat-recruitment GTPase.

The coat-recruitment GTPases also have a role in coat disassembly

Thus, a fully formed vesicle will be produced only when bud formation occurs faster
than the timed disassembly process
 Vesicle targeting involve two proteins

Specificity in targeting is ensured because all transport vesicles display surface markers that
identify them according to their origin and type of cargo, while target membranes display
complementary receptors that recognize the appropriate markers
This specificity is mediated by two proteins i) Rab proteins that direct the vesicles to
specific spots on the correct target membrane and ii) SNARE proteins that mediate the
fusion of the lipid bilayers

SNAREs, these transmembrane proteins exist as complementary setsvesicle membrane

SNAREs, called v-SNAREs, and target membrane SNAREs, called t-SNAREs
 Mechanism of vesicle docking

Rab effector proteins interact with the Rab proteins located on the target membrane,
vesicle membrane, or both to establish the first connection between the two membranes
Rab cascade : change in identity of the organelle
 SNAREs mediate membrane fusion

A v-SNARE is a single polypeptide chain, whereas a t-SNARE is composed of two or three

proteins. They form a four-helix bundle resulting trans-SNARE complexes lock the two
membranes together.

Best e.g. docking and fusion of synaptic vesicle at the nerve terminals plasma
membrane in the process of neurotransmitter release. Bacteria causing tetanus produces
toxin that cleaves SNARE proteins at the nerve terminal.
SNAREs mediate membrane fusion

The interacting helices wrap around each other to

pull the membrane faces together, while
simultaneously squeezing out water molecules from
the interface
Release of free SNAREs
 Viral Fusion Proteins and SNAREs May Use Similar Strategies

Virusessuch as human immunodeficiency virus (HIV), which causes AIDSbind to cell-

surface receptors, and then the viral and plasma membranes fuse

An exposure of HIV fusion protein to receptors on the target cell membrane, uncovers
previously buried hydrophobic regions. These regions, called fusion peptides, are observed
to then insert directly into the hydrophobic core of lipid bilayer of the target membrane
 Transport from the ER through the Golgi Apparatus

Proteins Leave the ER in COPII-coated Transport Vesicles

Cargo proteins display exit

(transport) signals on their
surface that are recognized by
complementary receptor
proteins that become trapped
in the budding vesicle by
interacting with components of
the COPII coat
Only Proteins That Are Properly Folded and Assembled Can Leave the ER

The proteins that are misfolded or incompletely assembled are retained in the ER, where
they are bound to chaperone proteins

Case of Cystic Fibrosis

 Transport from the ER to the Golgi Apparatus Is Mediated by Vesicular Tubular
Clusters

The structures formed when ER-derived vesicles fuse with one another are called vesicular
tubular clusters
The clusters are relatively short-lived because they quickly move along microtubules to the
Golgi apparatus, where they fuse and deliver their contents

The retrieval transport pathway : carry back to the ER resident proteins that have escaped, as
well as proteins that participated in the ER budding reaction to ER. Theses retrieval vesicles
are coated with COPI.
 The Retrieval Pathway to the ER Uses Sorting Signals

Two types of signal for retrieval: i) two lysines, followed by any two other amino
acids (KKXX), at the extreme C-terminal end of the ER membrane protein. ii)
Lys-Asp-Glu-Leu or similar sequence (KDEL sequence) in ER soluble protein
 The Golgi complex
Golgi complex is a major site of carbohydrate synthesis, as well as a sorting and dispatching
station for the products of the ER

It consists of a collection of flattened, membrane-enclosed cisternae, somewhat

resembling a stack of pancakes

Proteins and lipids enter the cis Golgi network in vesicular tubular clusters arriving from the ER
and exit from the trans Golgi network bound for the cell surface or another compartment.
Distribution of sugar processing enzymes
 Oligosaccharide Chains Are Processed in the Golgi Apparatus
Two broad classes of N-linked oligosaccharides, the complex oligosaccharides and the
high-mannose oligosaccharides, are found attached to mammalian glycoproteins
Oligosaccharide processing in the ER and the Golgi apparatus
Not only N-linked glycosylation but also O-linked glycosylation takes place

e.g. mucins, the glycoproteins in mucus secretion

 The Purpose of Glycosylation

They function to aid folding and the transport process

Make the folding intermediate soluble, thereby preventing their aggregation

Can limit the approach of other macromolecules to the protein surface. In this
way, for example, the presence of oligosaccharides tends to make a
glycoprotein more resistant to digestion by proteases

The oligosaccharides attached to some cell-surface proteins, for example, are

recognized by transmembrane lectins called selectins, which function in cell-
cell adhesion processes

Glycosylation can also have important regulatory roles in signaling pathway

Two models for explaining Transport Through the Golgi Apparatus

Vesicular transport model: the Golgi apparatus is a

relatively static structure, with its enzymes held in place,
while the molecules in transit are moved through the
cisternae in sequence, carried by transport vesicles

Cisternal maturation model: the Golgi is viewed as a

dynamic structure in which the cisternae themselves
move through the Golgi stack. The vesicular tubular
clusters that arrive from the ER fuse with one another
to become a cis Golgi network, and this network then
progressively matures to become a cis cisterna, then a
medial cisterna, and so on
Evidence of maturation model
Thank You