BIOM 3530 - Week 6

Unfolded Protein Response (UPR) How does UPR work? UPR activates three different types of signal pathways to enable the

ER to better handle protein translation and folding during ER stress

Unfolded Protein Response (UPR) Sensors:

IRE1, PERK1, ATF6

IRE1: Transmembrane protein kinase with

•cytoplasmic kinase and RNAse domains

IRE1: Binding of misfolded proteins in ER activates

kinase and RNAse domain

IRE1: Causes splicing of pre-mRNA that produces

unique transcription factor (TF)

IRE1: TF turns on genes that expand ER, increases

•protein folding capacity and increases protein degradation of misfolded proteins

PERK1: Transmembrane protein kinase that

•phosphorylates translation factors (inhibiting overall protein synthesis)

ATF6: Transmembrane transcription factor that is cleaved when

•activated in golgi and turns on genes that can increase protein folding

summary slide. All transmembrane domain. All have binding sites i

ER lumen. All 3 of those 3 things with red are all diff activated TF that go in and activate capacities of ER.

IRE1, PERK1, and ATF6 all cause the activation of genes to increase

protein-folding capacity of ER

chaperones trying to fold them properly, if they cant, bind to

receptors

chaperones trying to fold them properly, if they cant, bind to receptors, get activated by

phosphorylation

chaperones trying to fold them properly, if they cant, bind to receptors, get activated by phosphorylation, gets spliced, and mRNAN is not translated into

TF

chaperones trying to fold them properly, if they cant, bind to receptors, get activated by phosphorylation, gets spliced, and mRNA is not translated into TF. Get exported out into cytoplasm and the chaperones have signal binding to

SRP receptor mentioned before and now secreted as luminal proteins to increase refolding

If cell cant slow everything down, cell goes into

apoptosis

more rna is cytoplasm are mature rnas spliced in the

nucleus

in this case, cells have pre-rna floating around in cytoplasm just in case. It still has an intron in it so ribosomes don't bind to it. But if IRE1 active the mrna gets spliced together creating

active messenger that becomes TF that go into nucleus and turn on gene expression

They can increase protein folding, expanding ER, or increase protein degradation of misfolded proteins. All initiated by

IRE1, PERK1, ATF6,. Depends on which one the cell is using, Activation of any of them causes same effect of increasing ER activity.

Perk1 is transmembrane kinase, activated and phosphorates a

specific transcription factor

So there are 3 sensors embedded in the ER membrane that detect misfolded proteins (ER stress).

IRE1

-PERK

-ATF6

Normally, these sensors are inactive bc chaperone

GRP78 is bound to them

When misfolded proteins accumulate, GRP78 leaves the sensors and binds to the misfolded proteins instead.

-The sensors become

activated and trigger a diff pathway to fix the problem

IRE1 - is transmembrane protein with cytosolic kinase and RNAse domains.

-When the GRP78 goes to bind misfolded proteins of ER, the

kinase and RNAse domains become active

They splice pre-mRNA (mRNA exists in the cytoplam but contains

introns that prevents proper translation) and produde a TF.

The TF turns on genes to:

expand ER size, increase chaperone proteins, and increase protein degradation of misfolded proteins (ERAD)

PERK1 - transmembrane protein kinase

-when activated, phosphorates a

translation inhibitior factor

PERK1 - transmembrane protein kinase

-when activated, phosphorates a translation inhibitor factor

-causes overall protein synthesis to

decrease

-The ER is overloaded so cell needs to stop sending new proteins into the ER.

ATF6 - transmembrane transcription factor (TF)

-When ER stress occurs, ATF6 moves from

ER → golgi

Doesnt producing more chaperones increase stress? Not really bc theyre there to fix the

misfolded proteins

Expanding the ER is also done at the same time so you're increasing the

protein capacity.

once protein made in ER, some stay but

majority leave

this part of the membrane is tagged that its gonna receive cargo and snips off from

membrane to form its own vesicle. How does it know where to go?

membrane invaginates and snip off from membrane and create its own little vessicle called

vesicle budding.

A lot of info from donor vesicle has alr been donated to the membrane so it knows where to

sent the vesicle

All vesicles contain many proteins:

Integral membrane proteins (adapters and other binding proteins)

Cargo proteins

Coat proteins

all vesicles have 3 major families of proteins. Also called

adaptor or binding proteins. Cargo protein are what are being moved. The coat proteins are unique

a vesicle that's gonna be released, usually packed full of neurotransmitter. Sits at the membrane waiting for

fusion event to release all the neurotransmitter in.

Inside packed full of cargo. On surface is many

transmembrane proteins

There are many pumps too to create proton gradients through

active gradients

Glutamate is an ex of neurotransmitter that is

brought in.

red is active zone where vesicles will be released. A lot of vesicles getting ready to fuse and some ready at

active zone

The copy # of these proteins is very high. Very

complicated and highly regulated.

Brain- Pre-synaptic terminal, 300,000 proteins, 60

different types of proteins

vary between 150 to

20,000 copies each

Blue: retrieval pathway

(retrograde trafficking)

retrieval pathway(retrograde trafficking)-

early endosome, late endosome, and secretory vesicles to golgi, to ER, while early endosome can also send to cell exterior

Green: endocytic pathway,

cell exterior to early endosome, to late endosome, to lysosome

Red: biosynthetic/secretory pathway

-Er to Golgi, Golgi to cell exterior, secretory vesicles, late endosome, and early endosome.

-Then late endosome to lysosome and secretory vesicles to cell exterior

There is a constant flow of proteins throughout cell mediated by

vesicle trafficking

Golgi Apparatus/Complex- Consists of

flattened stacked membrane compartments (cisternae)

(4-6 cisternae per stack)

Golgi Apparatus/Complex- Specific orientation of stacks that

mature" over time

cis-face: entry face at

beginning of Golgi (near ER)

medial: sandwiched by

cis and trans cisternae

trans-face: exit face just prior to

leaving Golgi

Proteins enter

Cis Golgi Network (CGN)

Proteins enter Cis Golgi Network (CGN) and

Leave via the

Trans Golgi Network (TGN)

proteins enter by cis golgi and leave by trans golgi. some get deposited

deposited into golgi itself but its mostly its a transporatory passage

goes all the way through until it leaves as vesicle from

trans face

The green parts are the lumen. There's Golgi enzymes that are made in

ER, transported to golgi and stay there

Proteins are post-translationally modified in Golgi in various

sub compartments, What type of

post-translational modification?

Glycosylation

major one that occurs in the golgi called glycosylation where sugars are added to aa on the protein. They need to be glycolated to

function properly

If there are cytosolic enzymes floating in lumen and vesicles fusing between each cisternea. Those enzymes can also end up being trafficked. Enzymes in cis can be transfered to

another, but its fine bc they just get retrograde trafficked back.

What are coat proteins?

Proteins which "coat" or cover the vesicle

coat proteins 3 types:

Clathrin, COP I (Coat Protein I), and COP II

Vesicles can not form without

coat proteins

donor component- place

vesicle is leaving from

target component-

usually the golgi, donor wants to fuse to the target

Presence of a particular coat protein depends on

where the vesicles originate from

Clathrin: transport vesicles from

PM and between endosomal and Golgi compartments

COP1: bud off from

Golgi

COP2: Early transport

from ER

Clathrin is also in

endosomal and Golgi complexes

COP2 is on proteins that has just been

translated, leaving coated in COP2

each clathrin subunit is composed of

three large (heavy chains) and three small (light chains)

each clathrin subunit is composed of three large (heavy chains) and three small (light chains) that form

triskelion

triskelion form combination of

hexagons and pentagons (polyhedral cages)

Clathrin is attached to

adaptor molecules in cell mb

Clathrin coat forms from triskelion structure upon

endocytosis

Clathrin coat is shed prior to

vesicle fusion

Clathrin is essential for

vesicle formation

Clathrin is essential for vesicle formation but not necessary for

vesicle transport

•Clathrin coat forms from triskelion structure upon endocytosis, essential for endocytosis to occur, only for formation of

vesicle, after formation, clathrin falls apart so the vesicle knows where to go

light chains and heavy chains interact at

central core

adaptor proteins form a second coat around vesicle and serve to

•anchor cargo receptors into the vesicle

each type of adaptor recognizes

different receptors

clathrin coat is shed prior to

vesicle transport

1) Cargo molecules bind to

cytoplasmic domains

2) Mb bends due to

clathrin structure

3) Clathrin triskelion pulls away from plasma mb as mb-bending and fission proteins

wrap around neck, breaking it off with no leakage

4) Coated vesicle mb leaves mb unaltered, then

loses the clathrin coat and is naked for transport

What are key regulatory molecules of vesicle trafficking?

Phosphatidyl Inositol Phosphates (PIPs)

PIPs are hydrophobic and inserts into

mb and tags, which can be recognized by clathrin

PIP structure

6 carbon sugar carb, phosphate group, and fatty acid

Fatty acid is

hydrophobic and can insert into plasma mb

Sugar carb- any carbon can be phosphorylated, and is included in the

name of the PIP, ex PI (3, 4) P2

Kinase adds the

phosphate groups onto the sugar

Phosphatases take away

phosphate groups on the sugar

Distinct sets of phosphatases and kinases reside in different

vesicle populations to alter PI molecules

Different PIs bind

different proteins

PI(5)P can be in high concentration in

lipid rafts