Synthesis of intergral membrane proteins (proteins embedded in detail

And synthesis of proteins to be excreted

5'

Translated

By way of SRP receptor

(SRP leaves, SRP receptor dissociates)

Growing polypeptide is going to cross the membrane

N-glycosylation = Xferring small chains of sugars (oligosacharides) from attachment to lipid and move over to protein. From Dolichol to asparagine in the polypeptide.

Take a protein in lumen and attach certain polypeptides to a glycolipid anchor

2 Fatty Acids

Glycerol Phosphate

"Head group on phospholipid" (choline, serine, ethanolamine)

Extracellular material (plasma membrane, receptor,bound ECM + PMR) into cytosol via vesicle formation

*Lysosome

Bud from smooth ER

Vesicles can fuse to form a tubular cluster

Buds -> Vesicles -> Golgi

Series of membranous sacks connected by vesicles

Site for protein modification

Clathrin - endocytosis

COPI - Back to the Endoplasmic reticulum

COPII - Leaving ER

3 heavy + 3 light chains

Interactions between triskelions help form a bud/pit on plasma membrane

Eventually pit gets budded off into a vesicle (soccer ball)

Membrane receptor binds to cargo

Binds adaptin

By way of adaptin binds clathrin

Clathrin does its work

Dynamin wraps around and pinches off

- Once in cytosol uncoating occurs (requires chapperone and has ATPase activity)

- Clathrin gets recycled

- One subunit binds to GTP binding protein in membrane, and binds to specific membrane receptors

- Other subunit binds to above complex to form outer coat

Specific membrane proteins called SNARES

V Snare and T Snare

GTP binding proteins

Can exist in cytosol as GDP

In vesicle, Cytosolic ends bind to what?

and

ER ends bind to what?

Cytosolic ends bind to coat

ER ends bind to cargo

Vesicles

and

Movement of the entire sac

Binds to COP I

Binds to KDEL signal peptide on ER lumen proteins

Returns to ER

- Sorting of proteins and targeting to vesicle

Modifies N-Linked oligosaccharides on glycoprotiens

(glycosolation)

Via threonine (oligosaccharide added)

and in the golgi

Adding of sulfur groups to certain proteins (tyrosine) and carbohydrates (glycosotation)

In trans golgi

- Lysosomes

- Plasma membrane

-Secretory vesicles

- If the endocytosed material is a nutrient

- If it is a phagocytosed bacterium

- Products from autophagy (eating self)

- Damagedcellular organelles

Other fates for vesicles leaving trans golgi?

Aside from lysosomes

Constituitive secretion

Regulated secretion

(always happening, no regulation)

- Delivers newly synthesized membrane proteins and lipids to plasma membrane

- Default pathway

- No signal or receptor required

- Aggregation of proteins in the trans golgi

- Aggregates are segregated into vesicles

-When signal is recieved secretory vesicles move to plasma membrane and fuse to dump the contents

Vesicles leave trans golgi and are stored in cytosol (secretory granules)

- Release response to a signal

(Secretion) cleaved at time of secretion

(of secretory granules)

Endocytosis ___ Surface area of plasma membrane

Exocytosis ___ Surface Area of plasma membrane

Endocytosis - decreases surface area of plasma membrane

Exocytosis increases surface area of plasma membrane

Low Density lipoprotein

More cholesterol, less protein

Plasma membrane and they aggrigate in clathrin coated pits

LDL particles bind to these and become like vesicles, then dynamin pinches off and now LDL is in cytosol

It goes into cytosol

 vesicles fuse -> endosome -> lysosome

Becomes Free cholestorl, and this down regulates de novo (from scratch) synthesis of cholesterol.

Coat gold particles of various size with NLS (nuclear localization signal) and inject into cytosol.

time course of entry into nucleus

To localize a protein

If localyzation is known, use to visualize compartment to see how big it is

- Fine point of light and blocking non fine light

- Illumination via point source (LAZR BEEEMZ)

- Out of focus light is illiminated from the image

- Image at each focal plane

- 3D reconstruction via computer

converts 2d images into 3d images

because individual sections can be misleading

- Flood cell with substrates and when enzymes (in lysosomes/vesicles)  interact (needs an acidic environment) it'll for a precipitate

Colored product - light microscopy

Heavy metal - electron microscopy

- Used for separating various cellular comparments/components

Need to break cell open (lysis) but needs to be a gently lysis technique

Example would be cell lisate

Centrifugation!

Parts of the cell sediemnt according to size, shape, and density of organelle

And a progressive increase in speed is needed!

1,000x G 10 min

Remove solid

20,000x G 20 min

Remove solid

Rinse n Repeat!

Most dense pallete is found at first, least dense toward end

size and shape of particle determines rate of sedimentation

Uses a weak sucrose gradient

Don't spin long enough to separateeverything (this will separate by density)

Uses buoyant density

Steep gradient of sucrose or salt

Separates on basis of buoyant density

Downs gradient and stops when its on same position of sucrose density

1. Wait!

2. Fractionate the cell

Use mini giger counter to find which parts have radioactivity

Radioactive label, inject labeled protein into cell, wait then fractionate

Use SDS page (you know size because you made it)

Is protein smaller?

put an xray film over SDS page to find out

Insert a protein into a cell and wait

After a bit insert protease and that will destroy anything in the cytosol

Do SDS page after protease to see if size has changed

Breaks down proteins!

Protein enzyme

It allows the experiment to stop and restart the process of protein transport

Can stop add protease, see if protein is safe, then restart

Change temperature of a cell to stop then restart actions

Used in conjunction with protease protection

Exchanging signal sequences on a protein

or Adding a possible signal to a protein that is usually cytosolic and verify if it is actually a signal

Isolate the mutants and see where they are blocked along the path between ER and PM

Ex. Group 1 is stuck in ER then it might have no signal sequence or a mutation not allowing SRP binding

2 populations of golgi, a donor and acceptor

Donor golgi has a protein (label this with radioactive sugar) but no enzyme

Acceptor golgi has a glycosylation enzyme but no protein

Donor golgi will bud of a vesicle and travel to the acceptor golgi and from here you can look for radioactive glycosylation

Bind SRP and this stops translation

Signal recognition particle

N-glycosylation (adding a carbohydrate chain via dolachol)

N = asperagene

COP II Coating protein binds (ER -> Golgi)

Uses a V snare and T snare to transport (these need Rab protein and a tethering protein)

Binds to M6P Receptor in the trans golgi

The M6P receptor binds to clathrin on cytosolic side

Regulated secretion - package aggregates into a secretory granule (stored until release)

Default constuitive - package goes straight to plasma membrane

Membrane proteins are glysylated proteins

True or false

What coat is used for endocytosis?

(as well as golgi to vesicle)

Goes to a lysosome, LDL receptors get recycled

Gets broken down to free cholestrol in lysosome

Free cholesterol slows down smooth ER synthesis of cholesterol

No ribosomes

Site for budding off vesicles

Bound ribosomes (cytosolic side)

Integration of transmembrane proteins

Recieves proteins to be secreted

ER-Lumen specific proteins

How are free and bound ribosomes different?

5'

END TERMINUS

a signal peptide

Signal sequence (peptide) at NH2 terminus of the polypeptide

there is no ER so they are pushing proteins across plasma membrane -> Secretions

SecA - ATPase (atp provides energy)

Destinations

ER lumen

ER membrane transmembrane proteins

NH2 end in cytosol

NH2 end in ER lumen
                        NH2 + COOH ends in cytosol or in lumen