3.1 & 3.2

Sorting Dilemma in a Eukaryotic Cell

How do we make sure each protein is delivered to the right place?

Endomembrane System (pathway)

transport vesicles bud from one compartment and fuse w/ other to carry cargo
Ribosomes—> ER—> Golgi—> Endosome/ lyososome/ extracellular space

Vesicle

membrane bound bubble that the cell uses to move things around

• bud from donor membrane compartment and fuse to acceptor membrane compartment

First Sorting of Proteins

finish translation in cytosol or transport mRNA-transcription to wherever it needs to go first, then finish translation (must contain signal sequence)

Co-translational Transport

protein is moved into ER before translation is finished

Necessary vs Sufficient

necessary: if you were to remove/ mutate signal sequence, it would end up in the wrong place

sufficient: if you were to copy & paste signal sequence and put in a different cargo that normally doesn’t go to destination, it will go to that new destination

Rough ER vs Smooth ER

RER is studded w/ ribosomes that are actively translating

Polyribosome

“many ribosomes” attached to one mRNA transcript at one time

• on the outer surface of ER, aligned in loops & sprials

Where is protein translation completed?

• protein destined to function in cytosol are completed in cytosol

• protein destined to function in endomembrane system or secreted are completed in ER

Lysosome

contain a variety of digestive enzymes, only active under acidic conditions

Pancreatic Acinar Cells

Highly secretory cells found in pancreas

• produce large amounts of digestive enzymes that are transported to intestines

Key experimental techniques that founded our knowledge of endomembrane function

• autoradiography of fixed cells

• subcellular fractionation and cell-free systems

Pulse-Chase

Pulse: cells are exposed to a labeled compound that can be distinguished/ detected. labeled compound is introduced into the molecule/ pathway to be studies.

Chase: cells are then exposed to same compound but unlabeled for varying amounts of time such that only those molecules labeled during pulse can be tracked over time

Subcellular Fractionation and Cell-free protein synthesis (steps)

1. Lyse the cell- break open the cell to release its contents

2. Fractionation- isolate rough ER using differential centrifugation

3. Reconstitution- add protein synthesis machinery to enable translation

In Vivo

• occurs naturally within living cells

• involved full endogenous cellular machery

• context dependent-influenced by cellular conditions

In Vitro

• occurs outside a living cell (test tube, petri dish)

• use extracted components

• controlled environment

Functional polysomes —> stripped microsomes

functional polysomes: detached from rough microsomes using non ionic detergent

stripped microsomes: derived from rough microsomes by detaching polysomes using high salt