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
Protein Synthesis and Transport within the Cell

The Endomembrane System

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
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
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 αα sequence or
Attached oligosaccharide
Protein Synthesis and Transport within the Cell

The Endomembrane System

Autoradiography
Example: Acinar cells – synthesis of digestive enzymes
Protein Synthesis and Transport within the Cell
The Endomembrane System
Autoradiography
Cells overlayed with photographic film
Exposed to radioisotopes in the sample
Radiolabeled αα
Protein Synthesis and Transport within the Cell
The Endomembrane System
Autoradiography
Tissues – washed and transferred to unlabelled αα
“pulse-chase”
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
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
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
Protein Synthesis and Transport within the Cell

The Endomembrane System

SER functions
Skeletal muscle
Kidney tubules
Steriod producing cells
Liver
Detoxification
Glucose-6-phosphatase
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
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
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
Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis
Begins after a messenger RNA binds to a free
ribosome
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
Protein Synthesis and Transport within the Cell
RER functions – Protein Synthesis
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
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)
Protein Synthesis and Transport within the Cell
RER functions – Processing of proteins
Misfolded proteins
Transported to cytosol
Destroyed in proteasomes
Quality control