Protein targe ng to the Endoplasmic Re culum 1 2 Protein Targe ng to the ER Proteins des ned for the cytoplasm have no targe ng sequences this is the default pathway Larger proteins des ned for the nucleus have an NLS and are taken up fully folded via nuclear pores gated transport Proteins targeted for the Endoplasmic Re culum also have signal sequences and are translocated across the ER membrane into a topologically dis nct compartment transmembrane transport ER Func ons Site of synthesis of membrane proteins and proteins of the secretory and endocy c pathways Proteins of the endomembrane system ER Golgi lysosomes endosomes secretory vesicles Plasma membrane proteins Site of lipid synthesis Ca storage Enzyme storage in some cell types Detoxifying enzymes in liver cells Topologically equivalent compartments in the secretory and endocy c pathways The ER is an extensive dynamic structure Network re culum of membrane tubules and sheets that stretches from the nucleus to the outer periphery of the cell Constantly changing shape extending and retrac ng During cell division the en re network breaks down into vesicles that par on into daughter cells ER structure Two types of ER membranes can be seen in the electron microscope One appears studded with ribosomes Rough ER and tends to be near the nucleus The other does not have ribosomes Smooth ER and tends to be in the more peripheral regions of the cell McGraw Hill Higher Educa on RER Structure Lumen Ribosomes RER structure RER ribosomes are bound to the membrane because of the nascent chains new proteins they are making The proteins are being co transla onally inserted into the membrane not post transla onally The two types of ER membranes can be separated by gradient centrifuga on Break cell mechanically microsomes form vesicles containing the membrane components that the ER had Centrifuge in sucrose density gradient Rough microsomes dense due to ribosomes Smooth microsomes lighter due to lack of ribosomes The two types of ER membranes can be separated by gradient centrifuga on Microsomes EM Free and Bound Polyribosomes Protein transla on is slow mRNAs are oVen long Once a ribosome has moved away from the start point on the mRNA another ribosome can aWach Many ribosomes can aWach to a single mRNA in this way it is now a polyribosome or polysome Most cellular proteins are made on polysomes If an ER targe ng signal is present in the protein the ribosome and nascent chain dock to the membrane Bound polysome Free polysomes and bound polysomes appear iden cal except for localiza on and pep de being made Process is e cient because the mRNA is always tethered to the membrane where there are abundant ribosomes being recycled Membrane bound and free polyribosomes A signal sequence directs proteins to the ER The ER signal sequence a stretch of hydrophobic amino acids N terminal signal sequence for soluble and some membrane proteins a k a signal pep de start transfer sequence Internal sequences for many membrane proteins Necessary and su cient for ER membrane targe ng You only need to add this sequence to a protein and it will go to the ER membrane Leads to co transla onal transloca on across ER membrane N terminal start transfer sequences are usually cleaved by signal pep dase so the sequence is not found in the mature protein The protein folds in the lumen with the help of chaperones and other molecules This protein is now either soluble in the lumen of the ER or associated with the ER membrane usually C terminus Proteins des ned for the ER are transported by a cotransla onal transmembrane mechanism How do you demonstrate co transla onal transport Use protease protec on experiments to ask whether proteins are ever exposed Mix rough microsomes have ribosomes mRNA ATP tRNAs AAs wait for transla on to nish then add protease Proteins are synthesized and are protected from protease co or post Mix rough microsomes mRNA ATP etc protease simultaneously Proteins are synthesized and are protected from protease co Mix rough microsomes mRNA ATP etc protease and detergent Proteins are now degraded protease enters microsomes How do you demonstrate co transla onal transport Use protease protec on experiments to ask whether proteins are ever exposed Mix rough microsomes have ribosomes mRNA ATP tRNAs AAs wait for transla on to nish then add protease Proteins are synthesized and are protected from protease co or post Mix rough microsomes mRNA ATP etc protease simultaneously Proteins are synthesized and are protected from protease co Mix rough microsomes mRNA ATP etc protease and detergent Proteins are now degraded protease enters microsomes Conclusion The proteins are protected during their synthesis transport into the lumen of the membranes is during synthesis cotransla onal transloca on The proteins are protected aVer synthesis from protease by the membrane detergent will permeabilize membrane allowing degrada on Co transla onal import Mix rough microsomes mRNA and ATP then add protease after waiting High Molecular Weight Low Molecular Weight Mix rough microsomes mRNA and ATP in the presence of protease Mix rough microsomes mRNA and ATP in the presence of protease and detergent protein synthesized with just ribosomes ATP and mRNA Co transla onal import Mix rough microsomes mRNA and ATP then add protease after waiting High Molecular Weight Low Molecular Weight Mix rough microsomes mRNA and ATP in the presence of protease Mix rough microsomes mRNA and ATP in the presence of protease and detergent protein synthesized with just ribosomes ATP and mRNA Co transla onal import Mix rough microsomes mRNA and ATP then add protease after waiting Mix rough microsomes mRNA and ATP in the presence of protease Mix rough microsomes mRNA and ATP in the presence of protease and detergent protein synthesized with just ribosomes ATP and mRNA High Molecular Weight Slightly larger because no signal pep dase from microsome to cleave signal sequence Low Molecular Weight How is the ER targe ng signal recognized Signal sequence emerges from ribosome Associates with the Signal Recogni on Par cle SRP Signal Recogni on Par cle SRP recognizes the signal sequence SRP is found free in the cytoplasm Discovered as a factor that allowed aWachment of pure ribosomes to ER membranes SRP is a ribonucleoprotein par cle RNP RNA 6 proteins Binds to both the signal sequence and the ribosome Binding of the SRP to the nascent pep de chain halts transla on when the pep de is 70 100 AA long long enough to