© 2003 by default!slide 1 protein sorting, transport and modification part1 m. saifur rohman, md,...

© 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


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


Sorting of secreted proteins


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


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)


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)


Specific uptake-targeting sequences


Uptake-targeting sequences of imported mitochondrial proteins


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


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


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


The rough ER is an extensive interconnected series of flattened sacs


Secretory proteins are found in the ER lumen immediately

after synthesis


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


Analysis of yeast mutants defined the major steps in the secretory pathway


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


Translocation into the ER


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Post-translational Translocation into ER


After insertion into the ER membrane, some proteins are transferred to a GPI anchor


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


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


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


Addition of N-linked sugars in the ER


Formation of S-S bond by Protein Disulfide Isomerase (PDI)


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


Modification of Proteins - Proteolytic Cleavage

Proteolytic cleavage of proinsulin occurs in secretory vesicles (after Golgi)


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


Processing of N-linked glycoproteins in the Golgi apparatus

Mannose trimming

Gucosamine addition

Galactose addition + neuraminic acid linkage to galactose


Protein Transport between Organelles are done by Vesicles

Assembly of protein coat drives vesicle formation and selection of cargo molecules


Assembly and Disassembly of Coat protein

Interaction of cargo protein with vesicleN-terminus of Sar1 (membrane anchor) not shown


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


Involvement of the 3 major types of coat proteins in traffic and secretory pathways


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


Receptor-mediated endocytosis generally occurs via clathrin-coated pits and vesicles


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


Receptor-Mediated Endocytosis


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Receptor-Mediated Endocytosis

© 2003 by default!slide 1 protein sorting, transport and modification part1 m. saifur rohman, md,...

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