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 aa 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 aa

Protein Synthesis and Transport within the Cell The Endomembrane System Autoradiography Tissues – washed and transferred to unlabelled aa “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