Protein Targeting, Transmembrane Protein Transport, Co-Translational Translocation, SRP

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Biochemistry & Molecular Bio.
David Gross

March 24 • Protein targeting to the ER o protein targeting to ER is example for transmembrane transport, also applies to mitochondria, plastids (chloroplasts), and peroxisomes o ER is entry point for majority of luminal & integral membrane proteins of endomembrane system, the plasma membrane & for proteins secreted to the cell exterior • General themes for transmembrane protein transport o intrinsic targeting signal that’s specific for the organelle o targeting/sorting factor & molecular chaperone that guides new protein to organelle surface o membrane targeting receptor that specifically recognizes targeting/sorting factor & cargo o transport/translocation channel that mediates membrane transport while maintaining membrane seal o molecular motor that provides driving force for membrane translocation • Creating an assay of protein transport/import o protein transport/import can be assayed in vitro using isolated organelles or microsomes & in vitro translated proteins o process:  in vitro synthesis preprotein with radiolabelled amino acids  add organelle or microsomes, pick up radiolabelled protein • mitochondria, chloroplasts & peroxisomes can be isolated from cells intact & functional  centrifuge the cell, labeled protein co-fractionates w/ organelle during centrifugation  the signal sequence is then removed by a specific protease that is present inside the organelle  protein protected from digestion when proteases added to incubation medium, susceptible if a detergent added to disrupt organelle membrane • ER sub-regions o rough ER  protein synthesis  membrane synthesis o smooth ER  synthesis of lipids: oils, phospholipids & steroids  polysaccharide metabolism  regulation of calcium concentration  detoxification of drugs & toxins • Role of Rough ER in protein synthesis o the signal sequence for a protein is usually located @ the N-terminus & is usually cleaved off before (during) entry into ER lumen  exception is if the membrane protein contains an internal signal sequence o 2 major features distinguish transmembrane transport from gated transport  protein substrate is unfolded polypeptide  the membrane channel is sealed o process:  a protein with the specific signal sequen
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