rough-rough-Endoplasmatic Endoplasmatic ReticulumReticulumrERrER

Sorting Sorting ofof

proteinsproteins

László KŐHIDAI, Med. Habil. MD.,PhD. Assoc. Professor

Department of Genetics, Cell- andImmunobiology

Semmelweis UniversitySept 29/ 2014

Endoplasmic = inside the cell; reticulum = networkEndoplasmic = inside the cell; reticulum = network

• Extensive membrane systemExtensive membrane system• Includes up to half of membrane of cellIncludes up to half of membrane of cell• Tubules and sacs = cisternaeTubules and sacs = cisternae• Continous with the nuclear envelopeContinous with the nuclear envelope• Two types: Two types: rough ER (ribosomes)rough ER (ribosomes)

smooth ERsmooth ER

rERrER

s-ER (smooth ER)s-ER (smooth ER)

Structure: tubularStructure: tubular

Function:Function:• synthesis of phospholipids, cholesterol,synthesis of phospholipids, cholesterol,

ceramideceramide• synthesis of steroidssynthesis of steroids• storage and regulation of Castorage and regulation of Ca2+2+ • detoxification – cyt P450detoxification – cyt P450

TEM of ribosomes attached to the rER in TEM of ribosomes attached to the rER in a pancreatic exocrine cella pancreatic exocrine cell

mRNAmRNA

peptidepeptidepolyribosomepolyribosome

Ribosomes – mRNA – PolyribosomeRibosomes – mRNA – Polyribosome

Molecular composition of Molecular composition of ribosomeribosome

60S rRNA + peptides60S rRNA + peptides rRNArRNA

Ribosome subunitsRibosome subunits

Comparison of prokaryotic and Comparison of prokaryotic and eukaryotic ribosomeseukaryotic ribosomes

Structure of ribosomeStructure of ribosome ??

t-RNAt-RNA

activator activator enzymeenzyme of AAof AA

ribosomeribosome

anticodonanticodon

codoncodon

InitiationInitiation

ElongationElongation

Peptide bond formationPeptide bond formation

peptidyl transferase

peptide bond

TerminationTermination

Internalization of peptides into Internalization of peptides into the rERthe rER

Synthesis of secretory proteins Synthesis of secretory proteins on the rERon the rER

Structure of SRPStructure of SRP

• UniversalUniversal• 300 base RNA 300 base RNA • Six proteinsSix proteins• P54 - signal peptideP54 - signal peptide• P9, P14 - ribosomeP9, P14 - ribosome• P68, P72 move the P68, P72 move the peptidepeptide

Synthesis of secretory proteins Synthesis of secretory proteins on the rERon the rER

Electron microscopic view of a Electron microscopic view of a translocon channeltranslocon channel

The ribosome-translocon-ER membrane The ribosome-translocon-ER membrane complexcomplex

Translocon complexTranslocon complex• TRAMTRAM – (= translocating chain-associated membrane – (= translocating chain-associated membrane

protein) binds the protein) binds the signal sequencesignal sequence• Sec61pSec61p – major constituent of the translocon channel; – major constituent of the translocon channel; assembles into a assembles into a donut-like structuredonut-like structure•The The Sec 61 complexSec 61 complex binds the ribosomebinds the ribosome, ,

participates the transmembrane transferparticipates the transmembrane transfer

Cycles of GDP/GTDP exchange and GTP Cycles of GDP/GTDP exchange and GTP hydrolysis that drive insertion of nascent hydrolysis that drive insertion of nascent

secretory protein into the transloconsecretory protein into the translocon

Topologies of some integral membrane Topologies of some integral membrane proteins synthesized on the rERproteins synthesized on the rER

Synthesis and insertion into the ER membrane Synthesis and insertion into the ER membrane of the insulin receptor and similar proteinsof the insulin receptor and similar proteins

N-terminus faces to ER lumenN-terminus faces to ER lumen C-terminus faces to cytosolC-terminus faces to cytosol A signal sequence is cleavedA signal sequence is cleaved Stop-transfer membrane-anchor Stop-transfer membrane-anchor

signalsignal

Synthesis and insertion into the ER membrane of the Synthesis and insertion into the ER membrane of the asialoglycoprotein receptor and similar proteinsasialoglycoprotein receptor and similar proteins

C-terminus faces to ER lumenC-terminus faces to ER lumen

N-terminus faces to cytosolN-terminus faces to cytosol

No N-terminal ER signal sequenceNo N-terminal ER signal sequence

an uncleaved integral signal an uncleaved integral signal

membrane-anchor sequencemembrane-anchor sequence

Synthesis and insertion into the ER membrane Synthesis and insertion into the ER membrane of proteins with multiple transmembrane of proteins with multiple transmembrane

-helical segments-helical segments

- An uncleaved internal signal membrane-anchor sequence- An uncleaved internal signal membrane-anchor sequence- A stop-transfer membrane-anchor sequence- A stop-transfer membrane-anchor sequence- An uncleaved internal signal membrane-anchor sequence- An uncleaved internal signal membrane-anchor sequence Etc. Etc.

Post-translational modificationPost-translational modification• Proteolytic cleavage of proteinsProteolytic cleavage of proteins• GlycosilationGlycosilation

• AcylationAcylation• MethylationMethylation• PhosphorylationPhosphorylation• SulfationSulfation• PrenylationPrenylation• Vitamin C-dependent modificationsVitamin C-dependent modifications• Vitamin K-dependent modificationsVitamin K-dependent modifications• SelenoproteinsSelenoproteins

Proteolytic cleavageProteolytic cleavage

• Removal of signal peptide from Removal of signal peptide from preproproteinspreproproteinspreproteinspreproteins

• Signal peptidaseSignal peptidase

Properties of uptake-targeting signal sequences

Target organelleTarget organelle Usual signal Usual signal location within location within

proteinprotein

Signal Signal removalremoval

Nature of signalNature of signal

rERrER N-terminalN-terminal ++ „„core” of core” of 6-126-12 mostly mostly hydrophobichydrophobic amino acids, often amino acids, often proceeded by one or more basic proceeded by one or more basic

amino acidsamino acids

MitochondriumMitochondrium N-terminal N-terminal ++ 3-53-5 nonconsecutive nonconsecutive Arg or LysArg or Lys residues often with residues often with Ser and ThrSer and Thr; ;

no Glu or Aspno Glu or Asp

ChloroplastChloroplast N-terminalN-terminal ++ No common motives, generally No common motives, generally rich in Ser,Thrrich in Ser,Thr, , poor in Glu poor in Glu

and Aspand Asp

PerixisomePerixisome C-terminalC-terminal -- Ser-Lys-LeuSer-Lys-Leu

NucleusNucleus InternalInternal -- Cluster of 5 basic amino acidsCluster of 5 basic amino acids or two samller clusters separated or two samller clusters separated

by 10 amino acidsby 10 amino acids

GlycoproteinsGlycoproteinsPredominant sugars are: Predominant sugars are:

glucose, galactose, mannose, fucose,glucose, galactose, mannose, fucose, GalNAc, GlcNAc, NANAGalNAc, GlcNAc, NANA

O-glycosidic linkage – O-glycosidic linkage – hydroxyl group of Ser, Thr, hydrLyshydroxyl group of Ser, Thr, hydrLys

N-glycosidic linkage – consensus sequence N-glycosidic linkage – consensus sequence N-X-S(T)N-X-S(T)((BUT No PBUT No P))

Major N-linked families: Major N-linked families: high mannose type, high mannose type, hybride type, hybride type, complex type (sialic acids)complex type (sialic acids)

GlycosilationGlycosilation

rERrERN-linkage to GlcNAcN-linkage to GlcNAc

rERrER

O-linkage to GalNAcO-linkage to GalNAc

O-linked sugars:O-linked sugars:sugars coupled to UDP, GDP (mannose), CMP (NANA)sugars coupled to UDP, GDP (mannose), CMP (NANA)glycosprotein glycosylttransferaseglycosprotein glycosylttransferase

N-linked sugars:N-linked sugars:Requires a lipid intermediate Requires a lipid intermediate dolichol phosphatedolichol phosphate

N-GlycosilationN-Glycosilation

Glycosylphosphatodyl inositol (GPI) Glycosylphosphatodyl inositol (GPI) -anchored peptides-anchored peptides

GPI-anchoredGPI-anchoredpeptides becomepeptides becomethe outer surface ofthe outer surface ofthe surface membranethe surface membrane

Protein folding: Protein Disulfide Protein folding: Protein Disulfide Isomerase (PDI)Isomerase (PDI)

• Provides mechanism Provides mechanism for breaking incorrectlyfor breaking incorrectly paired disulfide bonds.paired disulfide bonds.

• The most stable foldedThe most stable folded sate is reachedsate is reached

Protein folding:Protein folding:

• Peptidyl-prolyl isomerase:Peptidyl-prolyl isomerase: accelerates accelerates rotationrotation about peptidyl-prolyl bonds about peptidyl-prolyl bonds

• Oligosaccharide protein transferase:Oligosaccharide protein transferase: transfers transfers carbohydrate chainscarbohydrate chains to the nascent to the nascentpolypeptide as they enter the lumen of ERpolypeptide as they enter the lumen of ER

• Calnexin, calreticulin: Calnexin, calreticulin: interact with interact with CHO groupsCHO groups of glycoproteins of glycoproteins

Protein signals:Protein signals:

• Integral, soluble proteins of ER, Golgi retrieved byIntegral, soluble proteins of ER, Golgi retrieved by the the KDEL-receptorsKDEL-receptors. They recognize the . They recognize the KDELKDEL signal signal (Lys-Asp-Glu-Leu at C-terminus).(Lys-Asp-Glu-Leu at C-terminus).

• ER membrane proteins have a ER membrane proteins have a KKXXKKXX (dilysine motif) (dilysine motif) on the C-terminus.on the C-terminus.

• Other ER membrane proteins possess di-arginine motif Other ER membrane proteins possess di-arginine motif on the N-terminus.on the N-terminus.

Chase-pulse techniqueChase-pulse technique

AntibioticsAntibioticsThey inhibit different steps of protein synthesisThey inhibit different steps of protein synthesis

• Actinomycin DActinomycin D - transcription (- transcription (complex with DNAcomplex with DNA))• RifamycinRifamycin - transcription (- transcription (RNA polymeraseRNA polymerase))• AmanitinAmanitin - transcription (- transcription (RNA polymerase IIRNA polymerase II))• StreptomycinStreptomycin - iniciation- iniciation• TetracyclineTetracycline - aminoacyl-tRNA - - aminoacyl-tRNA - A locusA locus interaction interaction• ErythromycinErythromycin - translocation of tRNA from - translocation of tRNA from A to P locusA to P locus• CycloheximideCycloheximide - - “ (only in eukaryotes)“ (only in eukaryotes)• ChloramphenicolChloramphenicol - - peptide bondpeptide bond formation formation• PuromycinPuromycin - termination- termination

Penicillins and Cephalosporins Penicillins and Cephalosporins - synthesis of bacterial cell wall- synthesis of bacterial cell wall (proteoglycans)(proteoglycans)

AAPA

actinomycinactinomycinrifamycinrifamycinamanitinamanitin streptomycinstreptomycin

tetracyclinetetracycline

chloramphenicolchloramphenicol

erythromycin, cycloheximideerythromycin, cycloheximidepuromycinpuromycin