© 2003 by default!slide 1 protein sorting, transport and modification part1 m. saifur rohman, md,...
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© 2003 By Default!Slide 1
Protein Sorting, Protein Sorting, Transport and Transport and modificationmodification
part1part1
M. Saifur Rohman, MD, PhD, FIHAM. Saifur Rohman, MD, PhD, FIHA
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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
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Major Protein sorting pathways
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Sorting of secreted proteins
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Paths of Protein Trafficking
Com pleted proteinv ia P o s ttra n s la tio na l Im p o rt
G o es to :Cytosol, Nucleus, M itochondria, P lastids, Peroxisom es
Plasm a Mem brane
Secretory Vesicles
Late Endosom es m a tu reto fo rm Lysosom es
Vesicles from Golgifu se w ith Early Endosom es
to fo rm Late Endosom es
G olgi Apparatusv ia V e s icu la r T ra n sp o rt
P ro te in is fu rth e r m od if ieda n d g oe s to :
Ribosom es w ith partia lly synthesized proteins:A ttach to Endoplasm ic Reticulum
P ro te in is im p orte d in to E R via C o tra n sla tion a l Im p o rtP ro te in is m od if ied in E R a n d g o es to :
Ribosom es in C yto so l
Nucleusm R N A p rod u cedb y tra n sc rip tion
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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 systemsAdapted from Schatz and Dobberstein, Science 271, 1519 (1996)
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Protein pathways Proteins released into cytosol
– Processed to contain specific Processed to contain specific uptake-targeting uptake-targeting sequences (sequences (later removed by proteases)later removed by proteases)
– Imported into mitochondrion, chloroplast, peroxisome, Imported into mitochondrion, chloroplast, peroxisome, nucleusnucleus• Mitochondria and chloroplasts contain organelle DNA, Mitochondria and chloroplasts contain organelle DNA,
which encodes organelle rRNAs and tRNA, but few which encodes organelle rRNAs and tRNA, but few organelle proteinsorganelle proteins
• Most mitochondrial and chloroplast proteins are Most mitochondrial and chloroplast proteins are encoded by nuclear genes, which are translated by encoded by nuclear genes, which are translated by cytosolic ribosomes and then importedcytosolic ribosomes and then imported
– May be sorted to May be sorted to other organellar compartments other organellar compartments (second signal sequence)(second signal sequence)
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Specific uptake-targeting sequences
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Uptake-targeting sequences of imported mitochondrial proteins
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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
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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
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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
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The rough ER is an extensive interconnected series of flattened sacs
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Secretory proteins are found in the ER lumen immediately
after synthesis
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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
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Analysis of yeast mutants defined the major steps in the secretory pathway
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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 hold3.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
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Synthesis of secretory proteins and their cotranslational translocation across the ER membrane
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Translocation into the ER
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Post-translational Translocation into ER
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After insertion into the ER membrane, some proteins are transferred to a GPI anchor
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Post-translational modifications and quality control in the rough ER
Newly synthesized polypeptides in the Newly synthesized polypeptides in the membrane and lumen of the ER membrane and lumen of the ER undergo five principal modificationsundergo five principal modifications
– Formation of disulfide bondsFormation of disulfide bonds– Proper foldingProper folding– Addition and processing of carbohydratesAddition and processing of carbohydrates– Specific proteolytic cleavagesSpecific proteolytic cleavages– Assembly into multimeric proteinsAssembly into multimeric proteins
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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
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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
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Addition of N-linked sugars in the ER
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Formation of S-S bond by Protein Disulfide Isomerase (PDI)
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Folding and assembly of MultimersHemagglutinin 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
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Modification of Proteins - Proteolytic Cleavage
Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)
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Golgi and post-Golgi protein sortingGolgi 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 processinglate in maturation
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Processing of N-linked glycoproteins in the Golgi apparatus
Mannose trimming
Gucosamine addition
Galactose addition + neuraminic acid linkage to galactose
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Protein Transport between Organelles are done by Vesicles
Assembly of protein coat drives vesicle formation and selection of cargo molecules
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Assembly and Disassembly of Coat protein
Interaction of cargo protein with vesicleN-terminus of Sar1 (membrane anchor) not shown
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Model for Docking and Fusion of Transport vesicles with Target Membrane
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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
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Involvement of the 3 major types of coat proteins in traffic and secretory pathways
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At least three types of coated vesicles transport proteins from organelle to
organelle
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The secretory and endocytic pathway of protein sorting
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Receptor-mediated endocytosis generally occurs via clathrin-coated pits and vesicles
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Clathrin Coats
© 2003 By Default!Slide 42The endocytic pathway The endocytic pathway
delivers transferrin-bound delivers transferrin-bound iron to cellsiron to cells
Transcytosis moves some Transcytosis moves some ligands across cellsligands across cells
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Receptor-Mediated Endocytosis
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Receptor-Mediated Endocytosis
© 2003 By Default!Slide 45The LDL receptor binds and internalizes cholesterol-
containing particles