BIO230 - Lecture 1 - Membrane trafficking

what is essential for multicellular organisms?

-cell polarization

polar

-different at either end

-ends are different on a cell in some way

(ex. apical domain and basolateral domain)

what are the 3 things polarized cells can do?

1. have different functions at different cell regions

2. define inside vs. outside

3. transmit signals from one end to the other

what are some examples of polarized cells?

-epithelial cells

-nerve cells

membrane trafficking

-moving molecules to different membranes

-can send different proteins to different domains

-determines where proteins end up

what are the 2 ways membrane trafficking can establish polarity?

1. exocytosis directly to the target domain

2. exocytosis to any domain then selective endocytosis followed by recycling to the target domain

what are the basic principals of membrane trafficking?

• some trafficking routes are polarized (most of the time)

• proteins are organized at sorting stations (2 sorting stations)

• different routes are balanced by retrieval pathways (stuff going forward and backward)

what are the 2 sorting stations of membrane trafficking (organizing proteins)?

1. the trans golgi network

2. the endosome

what are the 2 types of membrane trafficking pathways for exocytosis directly to the target domain?

1. constitutive secretion pathway

2. regulated secretion pathway

constitutive secretion pathway

-the default pathway

-most cargo moves through this

-always gets phospholipid (always adds phospholipid)

-specific signals do not seem to be required for this pathway

-transmembrane proteins are an option for this pathway

regulated secretion

-can release material in response to a signal

-this pathways vesicles are fully-formed but do no fuse with the plasma membrane until a signal is received (that boost of phospholipid is very useful

-transmembrane proteins are also an option for this type of secretion

-can deliver extra membrane material

what are the 3 ways we can rely on regulated secretion to deliver that extra boost of phospholipid?

1. cytokinesis → one cell divides into two

2. phagocytosis → engulfs a molecule, it then forms a vesicle around it, moves it in as it forms that membrane around

3. plasma membrane repair → if cell is damaged and there’s a leak

what can either constitutive or regulated secretion pathways release?

-they can release concentrated cargo

how is concentrated cargo developed?

-clathrin-coated vesicles can return membrane back to the golgi, this shrinks the vesicle and makes the cargo more concentrated

what are the 3 options for endocytosed proteins (for exocytosis to any domain then endocytosis followed by recycling to the target domain)?

1. recycling to the same domain of the plasma membrane

2. transcytosis to the other domain of the plasma membrane

3. degradation in the lysosome

transcytosis

-moves molecule/protein to the other side

what is an example of endocytosis?

-cholesterol uptake

• cholesterol is inside an LDL binds to a LDL receptor that attaches to a clathrin coat, which creates a vesicle endocytosed into the cytosol surrounded by clathrin coating

• then once its in the cell, the vesicle is uncoated and the LDL+ cholesterol fuses with an early endosome and the receptor is recycled back to the plasma membrane

• the early endosome turns to a late endosome then a endolysosome (a lysosome fuses with it) and then the late endosome degrades

• the cholesterol is then released and that free cholesterol undergoes transcytosis

what are the 3 types of general membrane changes during vesicle trafficking?

1. vesicle forms from the donor membrane into the cytoplasm

2. vesicle fusion

3. vesicle forms from a donor membrane away from the cytoplasm (ex. virus) **can only occur outside

vesicle fusion

-vesicle merges with a target membrane

vesicle fusion process

-SNARE proteins help to mediate vesicle fusion

-both t-SNAREs and v-SNAREs are required

-t-SNAREs and v-SNARES must be on opposite membranes

vesicle forms from a donor membrane away from the cytoplasm process

-ESCRT proteins can form vesicles away from the cytoplasm

-some viruses leave with a bit of membrane (not all though) (the virus leaves and takes some extracellular membrane with it)

-vesicles can form away from the cytoplasm into lumen or extracellular space

-vesicle formation machinery (ESCRT proteins) is in the cytoplasm to help the virus leave with extra membrane

ESCRT protein forming vesicles away from the cytoplasm process

• PI(3)P and ubiquitin on a transmembrane viral protein) activate ESCRT-0

• then passes the molecules to ESCRT-1 → ESCRT-2 → ESCRT-3

• ESCRT-3 then builds up around the membrane and forms those extracellular vesicles

what are some examples of vesicle formation into the cytoplasm? (2)

-COPII-mediated secretory vesicle formation at the ER

-clathrin-mediated endocytic vesicle formation

phosphoinositides (PIPs)

-label different membrane domains

-all of types of these share the same basic structure

-can be phosphorylated on the inositol sugar (added phosphorylation)

-are named by their phosphorylated groups

-are interconverted by kinases and phosphatases

-combines with Rabs to give membranes different identities

what do different membrane domains and compartments contain?

-they contain different lipids

-different types of phosphoinositides are found at different subcellular locations

what is the basic structure of PIPs

head group → inositol sugar

glycerophospholipid → phosphate group + glycerol + lipids

(**in this order → inositol sugar is attached to the phosphate group on its C1)

**the phosphate group is also not through phosphorylation, it’s always apart of the structure (not added separ

what can phosphoinositides also be called?

-it can also be called phosphatidylinositol

what is the carbon numbering on the inositol sugar?

-carbon 1 is located on the right connected to the phosphate group then you count every other carbon counter-clockwise (its a 6 carbon ring)

what carbons are the possible phosphorylation sites on the inositol sugar of PIPs?

-carbons 3, 4, and 5

how are PIPs named?

-after PI

→ the phosphorylation site positions are shown in brackets (ex. PI(3))

-after P

→ the total number of phosphorylation sites are shown as a subscripts (ex. PI(3,4)P₂)

PIP kinase

-phosphorylates PIPs

-every one of this exists

-used to convert PIPs

PIP phoshatase

-dephosphorylates PIPs

-everyone of this doesn’t exist (ex. PI(5) can’t be phosphorylated to get back to PI)

-used to convert PIPs

how does membrane trafficking work?

-different proteins to different PIPs, which move the protein

what is the process of PI(4,5)P₂ targeting clathrin coat assembly, specifically the adaptor protein?

1. the adaptor protein (AP) binds to the PI(4,5)P₂, which opens up AP

2. now that AP is open, it then binds to cargo receptors with endocytosis signals

3. which fully activates AP, so it can undergo its function in clathrin coat assembly

**the PI (4,5)P helps the inner coat proteins bind to the cargo receptors and then they will start to work

Rab GTPases

-are molecular switches that can direct vesicles

-there are many different types of these molecules on different target membranes

GEF

-guanine nucleotide exchange factor

-it exchanges GDP for GTP, to turn on the Rab GTPase (GTP binding)

GAP

-GTPase activating protein

-it activates GTPase, helps turn off Rab GTPase (GTP hydrolysis)

what happens when Rab is bound to GTP and GDP?

• when Rab is bound to GTP → its on and active

• when RAB is bound to GDP → its off and inactive

Rab5

-is located on early endosomes, plasma membrane, and clathrin-coated vesicles

-combines with PIPs to give membranes different identities

what is the process of Rab GTPases?

1. when bound to GDP its inactive/off, which signals GEF

2. GEF then exchanges GDP for GTP through GTP binding

3. turning Rab GTPase on/active

4. then to turn it off GAP activates GTPase through GTP hydrolysis (phosphate is removed)

5. this causes Rab GTPase to turn off/inactive again

what is the process of Rab5 and PIPs combining to give membranes different identities?

1. Rab5-GTP (once active) recruits PI 3-kinase

2. PI(3)P can then recruit more Rab5-GEF

3. more Rab5-GEF makes more active Rab5-GTP (positive feedback loop)

• Rab5-GDP is attached to GDI and a covalently attached lipid (which is hidden), so its off/inactive

• then Rab5-GEF activates it so the lipid anchor is exposed and goes in the membrane, to activate Rab5-GDP to Rab5-GTP and it release GDI

• then PI 3-kinase phosphorylates PI to get PI(3)P creating a positive feedback loop for more recruitment of Rab5-GEF

• overall, Rab5-GTP and PI(3)P help activate the tethering proteins

**example: early endosome

GDI

-GDP dissociation inhibitor

what is the process of Rabs and SNAREs working together in vesicle targeting and fusion, specifically Rabs on the target membrane?

-active Rabs help activate the tethering protein to dock the incoming vesicle sending it to the t-SNARE complex interacting with the v-SNARE on the incoming vesicle, which removes the Rab-GDP on the incoming vesicle and bind it to GDI, then the vesicle will fuse with the target membrane

how are ends different?

-by having different proteins on each end