Cellular Endomembranes and Protein Transport: Lecture 1

Where is mRNA synthesized?

nucleus

Where are proteins synthesized?

cytoplasm and endoplasmic reticulum

What is the direction of protein synthesis?

From N terminal to C terminal

What is Eukaryotic Protein Sorting?

the cellular process that delivers newly made proteins to their correct functional destinations inside or outside a eukaryotic cell.

What does signal based targeting do?

Takes newly synthesized proteins form the cytoplasm to organelles

What is vesicle-based trafficking/secretory pathway?

Taking newly synthesized proteins from the ER to their final destinations

What are the steps in the Secretory Pathway?

1. Import into ER

2. Folding and Glycosylation in ER

3. Vesicular Transport from ER to Golgi

4. Cisternal Progression Through Golgi (glycosylation/modification)

5. Vesicular sorting at trans-golgi network

6. Vesicular trafficking to final destination

What is a membrane-bound protein?

a protein attached to the cytoplasmic face of the ER and engaged in the synthesis of Secretory Proteins

What is a cystolic Free ribosome protein?

a protein not attached to any cellular membrane and is engaged in the synthesis of non-secretory proteins

What is the major difference between Smooth and Rough endoplasmic reticulum?

the presence or absence of ribosomes on their outer membranes, which dictates their distinct functional roles in the cell

Why is rough ER significant?

It plays a central role in the synthesis and export of proteins.

The rough texture allows binding to the membrane.

What is a microsome?

a fragment of endoplasmic reticulum and attached ribosomes obtained by the centrifugation of homogenized cells.

Rough microsomes and smooth microsomes can be purified by...

equilibrium density centrifugation:

- Rough = denser = at bottom

- smoother = less dense = up top

How do we know proteins are made inside the ER?

Experiment using Nascent/radioactive secretory cells and microsomes.

Microsomes treated with detergent and protease displayed digestion of the protein.

Microsomes treated with only protease showed no digestion of the protein.

Rough microsomes show that translation and translocation occur...

simultaneously (co-translational translocation)

What is Co-translational Translocation

proteins synthesized on membrane-bound ribosomes enter the ER DURING translation

What is a signal sequence?

a short stretch of amino acids at one end of the polypeptide that determines the location of a protein in a cell

What are the 4 factors that contribute to co-translational translocation?

Signal Recognition Particle (SRP)

SRP receptor

GTP hydrolysis

Translocon

What is a signal recognitional particle (SRP)?

adaptor that bridges the gap between the ribosome making the protein and the ER membrane where the protein needs to go.

Attaches to the signal peptide sequence and leads the ribosome to the ER.

what step in co-translational translocation required energy?

The conformational change of the translocon

What is a translocon?

A membrane channel through which polypeptide chains with a signal sequence are transport into the ER

Where is the signal sequence cleaved and by whom?

By signal peptidase within the ER lumen

Where is the signal sequence on the protein?

At the N terminus

What are the types of ER membrane proteins?

Type I

Type II

Type III

Tail-anchored protein

Type IV

GPI-linked protein

Which type of ER membrane protein is the only one with a cleavable N-terminal ER signal sequence?

Type I

Type I ER membrane proteins

Only type that cleaves the signal sequence.

"Nexo-C cyto"

SRP involved

what does "Nexo-C cyto" mean?

N-terminal is in ER lumen and C-terminal is in cytosol

What is an example of a type I ER membrane protein?

Insulin receptors

Type II ER membrane protein

Lacks cleavable N-terminal signal sequence.

Required a single internal hydrophobic signal-anchor sequence.

"Cexo-Ncyto"

SRP Involved

What does "Cexo-Ncyto" mean?

N-terminal is in cytosol and C-terminal is in ER lumen

What is an example of a type II ER membrane protein?

Transferrin Receptor

Type III ER membrane protein

Lacks cleavable N-terminal sequence.

Requires a single internal hydrophobic signal-anchor sequence.

New synthesized protein faces to cytosol.

"Nexo-C cyto"

SRP involved

What is an example of a Type III ER membrane protein?

cytochrom P450

Tail anchored ER membrane protein

C-terminal tail-anchored.

Doesn't require SRP/SRP receptor/Translocon

Needs Get3 (ATPase) to bind to C-terminal hydrophobic topogenic sequence.

Type IV ER membrane protein

A mutlipass protein.

SRP involved

N & C term will always end up on the same side because of even # of helices.

GPI-anchored ER membrane proteins

Initial steps are similar to type i

GPI is not located in the cytoplasm, instead it is located in the exoplasmic or ER lumen.

What are the 4 principle modifications to proteins before reaching their final destinations?

1. Glycosylation

2. Formation of disulfide bond to ER

3. Proper folding of polytide

4. Proteolytic cleavage in ER, Golgi, and vesicle