© 2003 By Default!Slide 1 Protein Sorting, Transport and modification part1 M. Saifur Rohman, MD, PhD, FIHA

© 2003 By Default!Slide 2 Learning Objectives Describe the various ways in which proteins can be sorted/translocated – –Co-translational translocation – –Post-translational translocation Know the different mechanisms by which proteins leave the cytosol and enter the: – –nucleus – –mitochondria – –ER – –Golgi network The role of signal sequences in this process The fate of protein whose sorting signal was mutated The secretory pathway

© 2003 By Default!Slide 3 3 Major Protein sorting pathways

© 2003 By Default!Slide 4 Sorting of secreted proteins

© 2003 By Default!Slide 5

© 2003 By Default!Slide 6 Protein translocation systems IM, inner membrane; IMS, inner membrane space; P, periplasm OM, outer membrane; TL, thylakoid lumen; TM, thylakoid membrane SecYEG, Sec61, TOM, TIM, TOC are protein subunits of the translocation systems Adapted from Schatz and Dobberstein, Science 271, 1519 (1996)

© 2003 By Default!Slide 7 Protein pathways Proteins released into cytosol –Processed to contain specific uptake-targeting sequences (later removed by proteases) –Imported into mitochondrion, chloroplast, peroxisome, nucleus Mitochondria and chloroplasts contain organelle DNA, which encodes organelle rRNAs and tRNA, but few organelle proteinsMitochondria and chloroplasts contain organelle DNA, which encodes organelle rRNAs and tRNA, but few organelle proteins Most mitochondrial and chloroplast proteins are encoded by nuclear genes, which are translated by cytosolic ribosomes and then importedMost mitochondrial and chloroplast proteins are encoded by nuclear genes, which are translated by cytosolic ribosomes and then imported –May be sorted to other organellar compartments (second signal sequence)

© 2003 By Default!Slide 8 Specific uptake-targeting sequences

© 2003 By Default!Slide 9 Uptake-targeting sequences of imported mitochondrial proteins

© 2003 By Default!Slide 10 Translocation into mitochondria  Cytosolic chaperones deliver proteins to channel-linked receptors in the mitochondrial membrane  Delivery of preproteins depend on Hsp70/Hsp40 or MSF  Uptake of mitochondrial proteins requires energy: ATP in the cytosol, the proton- motive force across the inner membrane, and ATP in the matrix

© 2003 By Default!Slide 11 Translocation into peroxisomes  Targeting of proteins is initiated post-translationally by Pex5/7 proteins, which bind the peroxisomal targeting signal (PTS), SKL  Translocon is Pex14p receptor  Gated pore that is regulated by membrane proteins?  First organelle demonstrated to import proteins without a PTS, by virtue of assembly with other proteins that contained a PTS  Various protein oligomers are imported into peroxisomes  Peroxisomal protein import is defective in some genetic diseases, e.g., Zellweger syndrome

© 2003 By Default!Slide 12 Protein pathways Secretory pathway – –Ribosome/mRNA/protein complexes are directed to rough endoplasmic reticulum by ER signal sequence – –After translation, proteins move via transport vesicles to Golgi apparatus – –Packaged (disulfide bonds, addition of carbohydrates, proteolytic cleavages, assembly into multimeric units) proteins directed to cell surface (secretion), lysosome, or plasma membrane

© 2003 By Default!Slide 13 The rough ER is an extensive interconnected series of flattened sacs

© 2003 By Default!Slide 14 Secretory proteins are found in the ER lumen immediately after synthesis

© 2003 By Default!Slide 15 Overview of the secretory pathway  Cisternal progression  New proteins in ER lumen or membrane incorporated into vesicles, which fuse with cis- Golgi or with each other  Migration from cis- to trans- Golgi (localization), and undergo modifications  Some remain, while others move via small vesicles to cell surface or lysosomes  Secretion may be regulated or constitutive

© 2003 By Default!Slide 16 Analysis of yeast mutants defined the major steps in the secretory pathway

© 2003 By Default!Slide 17 Synthesis of secretory proteins and their cotranslational translocation across the ER membrane What is needed for translocation: 1.Signal sequence (9-12 hydrophobic AA with some mainly pos. charged ones – in some prokaryotes sometimes longer, most of the times cleaved off by peptidases on the ER lumen side, sequence mainly at N-terminal) 2.Signal-Recognition-Particle (SRP) –recognizes signal sequence of ribosome complex (ribosome with mRNA), redirects ribosome complex to SRP receptor, puts synthesis of protein on hold 3.SRP receptor – binds the ribosome- SRP complex - driggers that ribosome complex is moved to translocon (GTP dependent) 4.Translocon is a protein channel, opens upon binding of ribosome complex, synthesis through channel

© 2003 By Default!Slide Synthesis of secretory proteins and their cotranslational translocation across the ER membrane

© 2003 By Default!Slide 19 Translocation into the ER

© 2003 By Default!Slide Post-translational Translocation into ER

© 2003 By Default!Slide 21 After insertion into the ER membrane, some proteins are transferred to a GPI anchor

© 2003 By Default!Slide 22 Post-translational modifications and quality control in the rough ER Newly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications Newly synthesized polypeptides in the membrane and lumen of the ER undergo five principal modifications –Formation of disulfide bonds –Proper folding –Addition and processing of carbohydrates –Specific proteolytic cleavages –Assembly into multimeric proteins

© 2003 By Default!Slide 23 Protein Modification Membrane and soluble secretary proteins synthesized on the ER have 4 possible modifications before the reach final destination: 1.Glycosylation in ER and Golgi 2.Formation of S-S bonds in ER 3.Proper folding and assembly of multisubunits in ER 4.Proteolytic cleavage in ER, Golgi, and secretory vesicles

© 2003 By Default!Slide 24 Protein Modification - Glycosylation O-linked glycosylkation: Attachment of sugars to OH of Ser and Thr Often contain only 1-4 sugar groups N-linked glycosylation: Attachment of sugars to amine N of Asn (Asn- X-Ser/Thr) Larger and more sugar groups -> more complex Glycosylation patters differ slightly between spieces !!! Precursor of N-linked sugars that are added to proteins in the ER

© 2003 By Default!Slide 25 Addition of N-linked sugars in the ER

© 2003 By Default!Slide 26 Formation of S-S bond by Protein Disulfide Isomerase (PDI)

© 2003 By Default!Slide 27 Folding and assembly of Multimers Hemagglutinin trimer folding Binding of Chaperone BiP Closing S-S bond, N-linked glycosylation Membrane anchoring Assembly of trimer Another example for assembly of multimers -> immunoglobulins

© 2003 By Default!Slide 28 Modification of Proteins - Proteolytic Cleavage Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)

© 2003 By Default!Slide 29 Golgi and post-Golgi protein sorting Sequences in the membrane-spanning domain cause the retention of proteins in the Golgi Different vesicles are used for constitutive and regulated protein secretion Proproteins undergo proteolytic processing late in maturation

© 2003 By Default!Slide 30 Processing of N-linked glycoproteins in the Golgi apparatus Mannose trimming Gucosamine addition Galactose addition + neuraminic acid linkage to galactose

© 2003 By Default!Slide 31 Protein Transport between Organelles are done by Vesicles Assembly of protein coat drives vesicle formation and selection of cargo molecules

© 2003 By Default!Slide 32 Assembly and Disassembly of Coat protein Interaction of cargo protein with vesicle N-terminus of Sar1 (membrane anchor) not shown

© 2003 By Default!Slide 33 Model for Docking and Fusion of Transport vesicles with Target Membrane

© 2003 By Default!Slide 34 34

© 2003 By Default!Slide 35 Vesicle-mediated Protein Trafficking between ER and Golgi Backtransport mainly used for: -> recycling of membrane bilayer -> recycling of proteins (SNARE) -> missorted proteins Normal transport of secretory proteins

© 2003 By Default!Slide 36 Involvement of the 3 major types of coat proteins in traffic and secretory pathways

© 2003 By Default!Slide 37 At least three types of coated vesicles transport proteins from organelle to organelle

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© 2003 By Default!Slide The secretory and endocytic pathway of protein sorting

© 2003 By Default!Slide 40 Receptor-mediated endocytosis generally occurs via clathrin-coated pits and vesicles

© 2003 By Default!Slide 41 Clathrin Coats

© 2003 By Default!Slide 42 The endocytic pathway delivers transferrin-bound iron to cells Transcytosis moves some ligands across cells

© 2003 By Default!Slide 43 Receptor-Mediated Endocytosis

© 2003 By Default!Slide Receptor-Mediated Endocytosis

© 2003 By Default!Slide 45 The LDL receptor binds and internalizes cholesterol- containing particles