Lecture 17: The endomembrane system, the ER and protein synthesis

Question 1

List the general functions of the endomembrane system

Answer 1

1. A series of discrete, dynamic
membrane bound spaces
including:
• Endoplasmic reticulum
• Golgi complex
• Endosomes
• Lysosomes
• Vacuoles (in plants)

2. Site of synthesis of many proteins and small molecules
   (e. g. hormones)
3. Responsible for the synthesis, sorting and transport of these proteins and small molecules to their appropriate target location withing the EM system and to outside the cell.

Question 2

endomembrane theory

Answer 2

hypothesizes that eukaryotic cells arose from an anaerobic archaea cell lineage.
The plasma membrane invaginated (increase in surface area!) and eventually became the nuclear envelope and parts of the endomembrane system to form an anaerobic eukaryotic cell.
Then later on the anaerobic eukaryotic cell ate an aerobic bacterium, which eventually became the mitochondria.

Question 3

Describe what (and how) materials are moved between EM compartments

Answer 3

Proteins and small molecules are transported between compartments of the EM system via vesicles
The cargo molecules are accumulated to a budding cite via
signals. The vesicle that is formed also has signals on it that tell the EM machinery where to send it.

Question 4

vesicles

Answer 4

small blebs of lipid bilayer membrane surrounding an aqueous interior.

Question 5

Vesicles can thus carry:

Answer 5

1. Soluble Proteins In Their Interior
2. Soluble Small Molecules In Their Interior
3. Integral Membrane Proteins In The Membrane Of The Vesicle
4. Membrane in the membrane of the vesicle…this is how other membranes can grow

Question 6

List the three major pathways in the EM system and describe what they do (in general)

Answer 6

Regulated secretion: (exocytosis) materials are stored in membrane-bound granules and are only released when the proper signal occurs. (e.g. neurotransmitter release, gastric juice release)

Constitutive secretion: (exocytosis) is a continual process (e.g. excreting extracellular matrix material)

Endocytic pathway: materials are brought into the cell or transferred from the Golgi to lysosomes. Note that this also brings in membrane from the PM.

Question 7

How are EM proteins made?

Answer 7

1. EM proteins are produced in the rough endoplasmic reticulum which is associated with the nuclear membrane
2. They are transferred via vesicles to the Golgi complex where they are modified and are sorted into different vesicles based upon targeting sequences

Question 8

Do all proteins get secreted?

Answer 8

Note that some of the proteins in the EM system are there to carry out functions within the EM system … not everything is secreted!

Question 9

. Compare and contrast the structural differences between rough and smooth ER
a. List things that occur in these compartments

Answer 9

Rough ER:
Series of flattened membrane sacs (cisternae)
with internal space (cisternal space or lumen) Associated with the nuclear membrane
Has many ribosomes: a site of protein synthesis (why we call it ‘rough’)
RER also synthesizes some small molecules and lipids

Smooth ER:
Highly curved and tubular membrane spaces Extends throughout the cytoplasm
Major site of lipid biosynthesis for making more membrane
Specialized site of some small molecule synthesis (e.g.. steroids)
Ca2+ sequestration in muscle cells (sarcoplasmic reticulum)

Question 10

Describe how different localization signals direct proteins to different compartments
a. Describe what happens to proteins that don’t have localization signals

Answer 10

Most protein localization is done through specific aa sequences that serve as signals to tell the cell where the protein goes.

1. Proteins made in the cytosol that have an NLS will be imported into the nucleus
2. Proteins made in the cytosol that have an other localization sequences (e.g. MtCh of ChPl) will be targeted to the appropriate organelle.
3. Proteins that have specific ER signal sequences tell the cell to make them on the RER and enter the endomembrane system. They can also have other sequences that dictate where they’ll go in the EM system

Proteins with no localization sequences just stay in the cytosol and do their job there!

Question 11

. Describe how ribosomes assemble and start translating mRNAs, and how they ‘know’ that a protein is
to go through the EM system

Answer 11

All ribosome subunits start as ‘free’ in the cytosol (i.e. not associated with the RER)
If the growing peptide doesn’t have a signal sequence, the polyribosome just stays in the cytosol as ‘free’

If there’s an N-terminal ER signal sequence, translation is paused and the ribosome is brought to the RER. Translation will continue through a pore in the RER membrane called the translocon. (the text calls this the ‘protein translocator’)
As a ribosome translates down the mRNA, the translocon moves too because the ER membrane is fluid. Thus polyribosomes can also form in RER-associated translation.

Question 12

7. List the steps in how a EM-resident soluble protein ends up in the RER
   a. Describe what happens at each step
   b. List the components that are G-proteins

Answer 12

1. Firsta‘free’ribosomestartstranslation
2. SRP(and RNA+protein hybrid)detect signal sequence and attachest SRP receptor on RER membrane (i.e. brings ribosome to RER). Both of the SRP and the SRP receptor are G-proteins.
3. SRP receptor binds to translocon.GTP is hydrolyzed which causes…
4. The signal sequence being loaded into the Translocon.This Opens The Translocon pore and functions as a “start” signal, allowing the rest of the protein to be threaded through it into the ER lumen as translation proceeds.
5. At the end translation,the C-terminus passes through the translocon.The Signal peptide is cut from the protein by a signal peptidase, freeing the soluble protein into the ER lumen. Chaperone proteins help it fold (which we’ll talk about later!).
6. The Cleaved Signal Peptide Somehow Gets Degraded.
   Note: the ER signal sequence can be at the N-terminus of a protein

Question 13

Describe how integral membrane proteins get ‘stitched’ into the RER membrane

Answer 13

1. The Transfer Starts As Normal
2. In The Middle Of The Polypeptide there’s a hydrophobic ‘stop- transfer’ sequence (which will
   ultimately form the alpha-helix of the TMD)
3. As soon as it the stop-transfer sequence is in the translocon,
   translation pauses. The TMD alpha-helix forms in the
   translocon
4. There’sa‘sidedoor’onthetransloconwhichopens,allowingthealphahelixTMDto diffuse into the membrane. Theribosomecontinuestranslating,buttherestoftheproteinismadeintothe cytosol side, so the C-terminus is cytosolic.

Question 14

Describe what determines whether the N- and C- termini of IM proteins will be either in the cytosol or in the ER lumen

Answer 14

1. If thestartsignalisattheverybeginning,theNterminusispassedintotheERlumen and the signal sequence is cleaved off. Thus, the N-terminus is in the lumen.
2. Iftheproteinendswithastartsignal,theC-terminusisintheERlumen. 3. Iftheproteinendswithastopsignal,theC-terminusisinthecytosol.

Question 15

List and describe the processes that can occur to proteins in the ER lumen

Answer 15

1. Protein folding:
2. Disulfide bond reshuffling:
3. Addition of extra chemical groups
4. Addition of lipids (to form lipoproteins)
5. Formation of multimeric proteins (quaternary structure of proteins)
6. Proteolytic cleavage
   - signal peptides are removed
7. Glycosylation

Answer 16

whenever there’s a start, the protein after the start goes into the ER.
So
If there’s a stop, the protein sequence protein after that stays in the cytosol.