Biology 1020 Unit 3: Intracellular Membranes and Protein Transport

endomembrane system

network of membranes in eukaryotic cells (nuclear envelope, ER, golgi, lysosomes, endosomes, plasma membrane) that work to modify, package, and transport proteins and lipids

• proteins can be secreted from a cell without having to cross another membrane or being exposed to the cytosol

topologically equivalent

essentially, a protein floating in the ER lumen can travel to the golgi, then to secretory vesicle and be dumped outside the cell without ever touching the cytoplasm

path that proteins follow

1. protein synthesis/translation begins in cytosol

2. if signal peptide sequence is present, protein is sent through endomembrane system, starting with ER (SRP Signal Recognition Particle drags ribosome to ER membrane)

3. ribosome moves to ER before translation has finished (translation and transportation happen at same time)

4. translation is completed and protein enters ER lumen

5. vesicle transport to golgi apparatus (cis side)

6. golgi processes them and vesicle buds off on trans side

7. protein goes to final destination: exterior (secretion), plasma membrane, lysosome, other parts of endomembrane system, etc

cotranslational translocation

protein is imported into an organelle (ER) at the same time it is being trnaslated

postranslational translocation

protein is completely synthesized and released by a ribosome into the cytosol before it is imported into its target organelle

necessary and sufficient

necessary: the information is needed/the result wouldn’t happen if it was removed. (ie. something happens to the “molecular address labels“ the protein would not go where it’s supposed to go)

sufficient: the material is all you need to get the desired result (ie. if you copy pasted the information to a random molecule, it would go to the destination)

rough ER

studded with ribosomes, site of cotranslational protein synthesis while the proteins are inserted into lumen. the proteins are then modified and sent through endoplasmic system

polysomes

many ribosomes attached to one mRNA transcript at a time. they are all translating mRNA transcript into protein —> many proteins! translation occurs from 5’ to 3’ end.

proteins destined to function in cytosol (or nucleus) are completed in….

the cytosol

proteins destined to function in endomembrane system or secreted are completed in …..

ER

golgi

• receives vesicles on cis side

• proteins move through golgi as modifications and further processing happens

• then protein leaves through trans side (closer to plasma membrane)

lysosomes

break down molecules and waste; contain variety of digestive enzymes, which are only active under acidic conditions —> H+ ions are pumped into lumen of lysosome acidic (against conc. gradient of H+)

zymogen granules

vesicles full of digestive enzymes to be secreted into the digestive tract lumen

key experimental techniques that founded knowledge of endomembrane function

• autoradiography of fixed cells: using radioactivity to label and detect newly synthesized proteins to track their path through time and where they eventually end up

• subcellular fractionation and cell-free systems

autoradiography of fixed cells

allows for the tracking of labeled molecules (DNA or proteins) within tissue sections over time

science behind autoradiography

carbon has unstable isotopes with extra neutrons that will undergo radioactive decay (and emit beta particle which we will track). we can use this to label molecules of interest that contain carbon.

1. released beta particle interacts w/ layer of gelatin containing silver hallide —> a silver metal spot called a grain is produced, which is how the presence and location of the labeled molecule within the cell is determined (you can see these grains with electron microscopy)

process of autoradiography

1. pulse: incubation for 3 minutes or very short time in radioactive amino acids. labels proteins being built at that exact moment

2. chase: radioactive precursor is washed away and replaced with nonradioactive resion —> labeled proteins can move through cells organelles

3. fixation: scientist fixes the cells at specific time intervals (this stops the chase)

4. cut cell into thin slices

5. add gel coating in the dark and let the specimen sit… during this time, the beta particles from radioactive decay will be released and react to form silver

6. use TEM

pulse chase experiments

pulse: cells exposed to trackable compound (ie. isotopes or fluorophores) for a bit; used to label the products created in that time period

chase

cells exposed to same not radioactive compound so that only molecules labeled during pulse can be tracked

what would happen without chase

all proteins that are newly synthesized would be labeled. if you stop incubation at different time points, you would eventually label the entire endomembrane system —> no idea which was the first step and what followed

with chase…

only tracking the cohort of proteins that was made during the 3 minute pulse window and seeing where they eventually end up with increasing durations of chase

Jamieson & Palade pulse chase experiment

• radioactive proteins appear first in ER

• then move from ER to golgi

• then move from golgi to secretory vesicles to be secreted from the cell

subcellular fractionation using differential centrifugation and cell free protein synthesis

cell free protein synthesis: studying protein synthesis or translation in a test tube after reconstituting system with purified parts

steps to prepare cell free system

1. lyse the cell to release its contents

2. fractionation: isolate RER (rough microsomes) using differential centrifugation

   1. further fractionation

      1. functional polysomes detached from rough microsomes using nonionic detergent (they are actively holding onto mRNA and growing protein chain into membrane) —> keeps protein together

      2. stripped microsomes derived by detaching polysomes using high salt —> destroys ribosomes

3. reconstitution: add protein synthesis machinery to enable translation in a test tube controlled environment (ie. ribosomes, mRNA, microsomes, promoters, etc)

rough microsomes

bits of rough ER after homogenization that still have polysomes attached

nascent

newly synthesized

how to remove obtain ribosomes and mRNA from rough microsome

use nonionic detergent treatment to solubilize membrane. ribosome is not denatured and protein chain remains attached to ribosome (retains functionality)

how to obtain stripped microsome

use high salt and protein synethsis inhibitor puromycin to interfere with interactions that keep ribosomes attached to membrane. this stops translation and causes ribosomes to fall off membrane. collect stripped microsomes by differential centrifugation. ribosome is no longer functional and nascent protein chain is released into membrane lumen

protein synthesis cocktail

extracted cytosol containing all the factors necessary for protein translation

• added to isolated rough microscomes or detached polyribosomes

• radioactive amino acids may be added to label newly synthesized proteins and detect using autoradiography