Lecture 03, Sept 11, 2017 (Chris Loewen)

Cell Structure II -Trafficking

3rd edition: Ch2, pp28 – 36
4th edition: Ch2, pp33 – 40

Golgi Controversy (See Lecture 02 summary)

- Vesicular transport model vs. cisternal maturation model

Vesicular Transport
- Different coat proteins (eg. COP I, COP II, Clathrin) that drive vesicle formation are used in
different cellular compartments. This process requires energy because membranes are in their
low energy state when they are flat.
- Coat proteins form spherical "cages" that recruit the cargo and drive formation of vesicles.
Vesicle formation requires energy and is initiated by GTP-bound GTPases.
- Understand the processes of:
A) Initiation & cargo selection & budding & scission: Generation of vesicles at the 'donor'
location (i.e. where the vesicle forms) by: initiation, cargo selection and SNARE integration,
budding and scission (i.e. formation and the releasing of the vesicle from the donor
membrane).
B) Uncoating: Uncoating of the membrane-enclosed vesicle after it is formed and released
into cytoplasm and the recycling of coat proteins back to the 'donor' membrane (this is driven
by GTP hydrolysis to GDP and coat disassembly. This is critical so that the cell doesn't have
to make new coat proteins every time a vesicle is formed. Coat proteins are recycled and the
GDP is exchanged for GTP.
C) Tethering: Targeting of vesicle to a specific 'acceptor' membrane location (where the
vesicle ends up) by tethering, docking and membrane fusion
o Tethering is initiated by Rab GTPases.
D) Docking & fusion: Docking and membrane fusion is initiated by SNAREs (alpha helical
membrane proteins) which are located on the surface of vesicles (vSNARE; 1 protein per
complex) and the acceptor 'target' membranes (tSNAREs; 3 proteins per complex). They
wind together to form a very stable 'coiled-coil' structure that brings the two membranes (i.e.
in 'trans') into very, very close proximity which, together with the energy generated in the
winding, facilitates the fusion of the vesicle and acceptor membranes (once this occurs the 't'
and 'v' SNAREs are in the same membrane (i.e. in 'cis'). Upon completion of the process,
'unwinding' proteins then dissociate the SNAREs in the complex so that they can be recycled
for future rounds of fusion.

Membrane Traffic
- Understand that 'exocytosis', which is the process of adding membranous material to the
plasma membrane (ie. by vesicle fusion). This process is balanced by 'endocytosis' which
removes membranous material from the plasma membrane back into the cell (i.e. by vesicle
budding from the plasma membrane back into the cytoplasm)
- Know that there are three types of endocytosis as we will be returning to these processes in
various tissues and organ systems later in the course:
- Phagocytosis: for large particles, like macrophages ingesting bacteria
- Pinocytosis: fluid, uses small vesicles; accounts for most of the endocytosis in most cells
 - Receptor-mediated: for cell-signaling, mediated by clathrin coats
- Understand trafficking/sorting of proteins using the specific example of trafficking of
lysosomal enzymes to the lysosome, an acidic (pH = 5) organelle that's vital to breaking
down and recycling cellular components:
1. Lysosomal enzyme (= cargo molecule), which needs to be trafficked/sorted to the
lysosome, is phosphorylated on mannose to generate 'mannose-6-phosphate' (M6P)
in the cis-Golgi.
2. M6P binds to the 'M6P Receptor' in the trans-Golgi (neutral, ~pH7), which
recruits the coat protein clathrin.
3. A vesicle containing the enzyme-receptor complex buds and is released by
scission from the trans-Golgi.
4. The vesicle uncoats and is then targeted to and fuses with the late endosome.
5/6. The acidic pH of the late endosome (acidic, ~pH5.5) releases the lysosomal
enzyme from the M6P receptor.
7/8. The free M6P receptor is then recycled back to trans-Golgi, while the lysosomal
enzyme is dephosphorylated and traffics to the lysosome where it now becomes
active (i.e. due to the even lower pH in the lysosome as well as the
dephosphorylation; this ensures that the enzyme is NOT active earlier during
trafficking from ER and Golgi).
Please note: Retromers are multi-protein coat complexes that help recycle cargo-laden vesicles
from endosomes back to the TGN (you do not need to know any of the identities of the specific
proteins shown in slide #38).

- Understand receptor-mediated endocytosis is a type of trafficking from the plasma membrane

back to the lysosome, the Golgi, or the plasma membrane itself. One specific example (of
many) is the receptor-mediated endocytosis of a secreted form of lysosomal enzyme that is
phosphorylated on mannose (i.e. as occurs when the enzyme is mistargeted to the plasma
membrane and released).
1. The secreted lysosomal enzyme binds M6P receptor on the plasma membrane.
2. The enzyme-receptor complex recruits clathrin and an endocytic vesicle forms
that buds into the cytoplasm
3/4. The vesicle uncoats and fuses with an early endosome, which then fuses with a
late endosome.
5. The acidic pH of the late endosome releases the lysosomal enzyme from the
M6P receptor and dephosphorylates the enzyme itself.
6/7/8. The M6P receptor then recycles back to either trans-Golgi or the plasma
membrane; the dephosphorylated, active lysosomal enzyme is trafficked to the
lysosome.

Lysosomes
- Understand the structure and transmission E.M. appearance of lysosomes and residual bodies.
- Understand the functions of lysosomes which have an acidic pH due to proton pumps in the
lysosomal membrane (ie. breaking down and recycling of cellular components as well as the
initiation of 'autophagy'=digestion of cellular organelles to provide cellular building blocks for
cell function and survival under extreme conditions)
 Clinical relevance:
- Tay-Sachs disease:
• Doesn't have the Hexosaminidase A (Hex-A) enzyme, which acts to break down specific
types of lipid (eg. ganglioside GM2 that forms multilayered membranes in the lysosomes).
Therefore, these lipids accumulate abnormally in lysosomes to toxic levels, especially in
nerve cells in the brain.
- Xanthomas:
• Occurs in patients with a deficiency in Low Density Lipoprotein receptors (LDL normally
carries the lipid cholesterol in blood). This most often occurs because the LDL receptor is
not trafficked to/recycled from the plasma membrane correctly.
• Because of this deficiency, cells can't take in LDL and can't catabolize cholesterol,
resulting in high levels of circulating cholesterol in the blood (familial
hypercholesterolemia) and lipid/cholesterol deposits/swellings in the skin = xanthomas;
also develop atheroschlerotic plaques in blood vessels leading to cardiovascular disease
(will be discussed in 18Lect, Circulatory System).

Lipid Traffic
- Lipids must be carefully trafficked around the cell as they are hydrophobic and not soluble in the
aqueous cytoplasm (ie. they cannot diffuse through the cell on their own 'unassisted' given that
oil/lipids and water/cytoplasm do not mix)
- Lipids can be trafficked either:
1) In vesicles (ie. within the bilayered membranes of vesicles that directly fuse, as described
above, with other vesicles, with other membrane-bound organelles, or with the plasma
membrane).
2) In a non-vesicular manner hidden within lipid transfer proteins (LTP's).
3) ERJs (lipid traffic that occurs at ER junctions AKA membrane contact sites)

- Classes of lipids (all have hydrophobic and hydrophilic regions):

o Glycerolipids: has a glycerol backbone, and a hydrophilic choline as well as two hydrophobic
fatty acids (membrane lipid)
o Sphingolipids: has a structurally different sphingosine instead of a glycerol backbone; some
still have a choline attached like glycerolipids, while others have a sugar attached; as well as
one hydrophobic fatty acid (make membranes more rigid)
o Sterols: cholesterol-based, alter the fluidity of membranes when inserted into them (control
melting point of membranes)
- During non-vesicular traffic, which does not require energy, lipids move down a
concentration gradient. This process is important for synthesis of lipids given that individual
enzymes involved in generating lipid precursors are in different organelles. The lipids are
transported from one membrane to another across a cytoplasmic gap at defined sites called ER
junctions (eg. between the ER and mitochondria for phospholipid synthesis to generate
phosphatidyl choline (PC) from phosphatidyl serine (PS) and phosphatidyl ethanolamine (PE);
also occurs at sites of cholesterol and sphingolipids synthesis).
- Lipid transfer via LTP: Non-vesicular lipids are transported inside LTP's, which have a
hydrophobic pocket/barrel to hide the lipid from the aqueous cytoplasm (i.e. looks like a
 clenched fist with the lipid barrier inside the fist with the thumbs as the lid). Transfer from
donor to acceptor membranes happens in a series of steps:
1. LTP binds to targeting receptors on donor membrane and loads cholesterol into the
barrel of the LTP.
2. LTP dissociates from the donor membrane and diffuses to the acceptor membrane.
4. LTP binds the acceptor membrane via targeting receptors and unloads the cholesterol
into the acceptor membrane.
5. LTP dissociates from the acceptor membrane so that it can used to transport another
lipid
- Know that there are many families of LTP's, which carry specific lipids to and from various
structures in the cell, either for transport or for lipid synthesis/modification.

Please note: you DO NOT need to know the specific names of the LTP's listed on Slide 47.

- Lipid transfer at ERJs: Non-vesicular lipid transfer often occurs at sites where the ER comes into
very close proximity with the membranes of other structures. These sites are stable and known as
ER Junctions (ERJ's).
- ERJs are formed by 'bridging complex' proteins that bind to each other to bring the membranes
of the ER and the neighbouring structures into very close proximity with each other (i.e. within
10nm but they do not fuse). This makes it possible for the LTP's to bind to receptors in both the
donor and acceptor membranes simultaneously which facilitates the efficient transfer of the lipid
between the two membranes (slide 57 where Phosphatidyl serine (PS) is being transferred from
the ER to the mitochondria where it is then converted by a mitochondrial enzyme complex to
generate Phosphatidyl ethanolamine (PE) as shown in slide 44).

Please note: ERJs are found in many different cell types (including yeast and plants) and they
can have additional functions such as efficient transport of calcium at ER junctions that form the
'Triad Junction' in skeletal muscle cells.