1. Protein Synthesis and Transport within the Cell
The Endomembrane System
Membrane organelles – a dynamic, integrated network
Materials are shuttled – small transport vesicles
Bud from a donor membrane compartment
Move in a directed manner
Pulled by motor proteins
Tracks – microtubules of the cytoskeleton
At their destination – fuse with membrane of acceptor compartment
Repeated cycles of budding and fusion

2. Protein Synthesis and Transport within the Cell
The Endomembrane System

3. Protein Synthesis and Transport within the Cell
The Endomembrane System
Typical biosynthetic pathway
Proteins synthesised in the endoplasmic reticulum
Modified through the Golgi complex
Transported to destination
Also a secretory pathway

4. Protein Synthesis and Transport within the Cell
The Endomembrane System
Secretory activities – two types
Constituitive
Transported from site of synthesis and secreted continuously
Regulated
Materials to be secreted are stored in large, densely packed, membrane-bound secretory granules
Discharged following specific stimulus
Examples: endocrine cells, acinar cells, nerve cells

5. Protein Synthesis and Transport within the Cell
The Endomembrane System
Materials moved out by secretory pathways
Materials moved in by endocyte pathway
From outside the cells to endosomes / lysosomes
Secreted Proteins – targeted by sorting signals
In the aa sequence or
Attached oligosaccharide

6. Protein Synthesis and Transport within the Cell
The Endomembrane System
Autoradiography
Example: Acinar cells – synthesis of digestive enzymes

7. Protein Synthesis and Transport within the Cell
The Endomembrane System
Autoradiography
Cells overlayed with photographic film
Exposed to radioisotopes in the sample
Radiolabeled aa

8. Protein Synthesis and Transport within the Cell
The Endomembrane System
Autoradiography
Tissues – washed and transferred to unlabelled aa
“pulse-chase”

9. Protein Synthesis and Transport within the Cell
The Endomembrane System
Biochemical analysis of subcellular fractions
Technique to homogenize cells
Cytoplasmic membranes fragmented and form vesicles
Types separated using cell fractionation
ER and Golgi – form microsomes
Fractioned into rough and smooth

10. Protein Synthesis and Transport within the Cell
The Endomembrane System
The Endoplasmic Reticulum (ER)
Two categories
Rough (RER)
Smooth (SER)
Composition of luminal or cisternal space unique
RER – ribosomes bound to cytosolic surface
SER – no ribosomes
RER & SER – many complex differences

11. Protein Synthesis and Transport within the Cell
The Endomembrane System
RER vs SER
RER
Extensive
Interconnected sacs – cisternae
Continuous with nuclear envelope
SER
Tubular
Interconnecting pipelines

12. Protein Synthesis and Transport within the Cell
The Endomembrane System
SER functions
Skeletal muscle
Kidney tubules
Steriod producing cells
Liver
Detoxification
Glucose-6-phosphatase

13. Protein Synthesis and Transport within the Cell
The Endomembrane System
RER functions – Protein Synthesis
Polypeptides synthesized at two locales
1. On ribosomes attached to cytosolic surface of RER
Proteins secreted from the cell
Integral membrane proteins
Soluble proteins within ER, Golgi, lysosomes, endosomes

14. Protein Synthesis and Transport within the Cell
The Endomembrane System
RER functions – Protein Synthesis
Polypeptides synthesized at two locales
2. Other polypeptides – on free ribosomes
Released into the cytosol
Enzymes
Peripheral proteins – plasma membranes
Proteins nucleus
Protein mitochondria, peroxisomes

15. Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis
How are proteins synthesized at different sites?
Secretory proteins – signal sequence
Polypeptide moves to cisternal space of ER
cotranslationally

16. Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis
Begins after a messenger RNA binds to a free ribosome

17. Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis
Other points to note
Interaction between signal sequence and SRP – a key step
SRP is the tag – enables specific binding of the complex to the ER
Binding through at least two interactions
SRP – SRP receptor
Ribosome and translocon
Following binding
Nascent peptide released from SRP
Inserted into translocon
On termination
Ribsome released
Translocon channel reverts to narrow conformation

18. Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis

19. Protein Synthesis and Transport within the Cell
RER functions – Processing of proteins
Polypeptide is moved from the translocon to the cisterna
Enzymes in the membrane
Signal peptide removed – signal peptidase
Carbohydrates added – oligosaccharyltransferase

20. Protein Synthesis and Transport within the Cell
RER functions – Processing of proteins
In the lumen
Molecular chaperones – BiP (binding protein)
Recognize and bind unfolded proteins
Aid in attaining native conformation
Movement of the protein into the ER lumen
Protein disulfide isomerase (PDI)

21. Protein Synthesis and Transport within the Cell
RER functions – Processing of proteins
Misfolded proteins
Transported to cytosol
Destroyed in proteasomes
Quality control