L16 - Protein Delivery

overview

• fwd transport pathway using transport vesicles is the default pathway

Protein Synthesis at ER (fwd pathway)

1. in nucleus: ribosome reads mRNA → get signal peptide to go to ER

1. ribosome attaches to ER protein complex on ER membrane

2. signal seq enters complex & stays, while the growing polypeptide emerges from ribosome + enters the ER lumen

1. mature & soluble proteins stays in ER lumen & the signal peptide is cleaved at membrane

uses chaperone proteins = small proteins that help other proteins achieve + maintain proper shape

• small & soluble & within ER lumen

• these ER lumen proteins may be packaged into transport vesicles leaving the ER bc they’re small & soluble

ex. help maintain a proteins shape at higher temps (heat shock proteins/Hsp)

→ discovered by fruit flies exposed to heat

ex. BiP = loading control & chaperone protein that work sin the ER lumen

Will any ER membrane proteins be packaged into transport vesicles?

No, they are too large to fit

Will any ER lumen proteins be packaged into transport vesicles?

ex. chaperon proteins like BiP

Yes, they are small & soluble

Golgi apparatus

= modify & distribute proteins made in ER

• each compartment has diff enzymes

→ to view these Golgi, u can use techniques to fluoresce/mark the enzyme within the Golgi

Structure

• cis face = receiving side from ER

• trans face = shipping side to plasma membrane

• has diff levels of flattened vacuoles

→ EGN = enter (cis Golgi network)

→ TEN = exit (trans Golgi network)

Animal cells : have ONE Golgi apparatus

ex. looking at a fibroblast (skin cell) using DIC + wide field fluorescence to see Golgi w antibodies against the Golgi enzyme

Plant cells: have MANY little Golgi

ex. looking at a plant cell using a fluorescent microscope with a fusion protein (Golgi enzyme : GFP)

How was this enzyme labelled?

a). enzyme: GFP fusion protein

b). antibodies - fluorescent dye

c). antibodies - gold ball

d). coomassie blue

• looking at an enzyme within the Golgi apparatus

c). antibodies - gold ball

Procedure:

why the others dont work:

Protein Transport: The Retrieval Pathway

Retrieval Pathway = return things from Golgi BACK to ER

ex. returning BiP (a small chaperone protein) to the ER lumen - via KDEL signal + KDEL receptors

VIA:

• KDEL signal = return to ER lumen

→ ONE directional

ex. BiP have KDEL signal

• KDEL receptor = high affinity for cargo in the Golgi (bc of lower pH) & low affinity in ER (bc of higher pH)

  

Protein Transport: Forward vs. Retrieval Pathway Summary

Protein Delivery: Experiments

Experiment 1: Remove KDEL sequence from BiPs

result : modified BiPs exported from the cell (thus retrieval DIDNT occur)

conclusion: KDEL sequence is NECESSARY for retrieval

Experiment 2: KDEL sequence is added to insulin

result : modified insulins cycle b/w Golgi and ER (thus added BiP with KDEL results in retrieval)

conclusion: KDEL sequence is SUFFICIENT for retrieval

Lysosomes/endosomes

= contain digestive enzymes that can breakdown ANYTHING

• acidic - bc of ATP-powered proton pumps

Delivery of Enzymes to Lysosomes

via: M6P signal

• ONE-way movement (Golgi→ lysosome)

• M6P receptor = high affinity for cargo in Golgi (high pH), low in lysosome

  

Mechanism:

• enzymes going to lysosome are given M6P signal in Golgi

2 steps:

→ allowed by diff. levels of the golgi

1. cis cisterna : UDP → UMP

2. medial cisterna : M6P attached

Delivery of Substrates to Lysosomes

substrates from plasma membrane → lysosomes

• vesicles formed by RME & phagocytosis at the plasma membrane fuse with lysosomes

ex. trasferrin (iron utilization) : imported into future RBCs with RME

Mechanism:

Which of these vesicle contains M6P receptors?

1 & 2

bc: M6P receptors are for digestive enzymes

Which of these vesicle contains Transferrin receptors?

3 & 4

Transport of digested materials into cytosol

• done w membrane transport proteins

ex. DMT1 transports iron

Examinable Content