Post-Translational Events I Protein Trafficking Translocation of Newly Synthesized Proteins into Membranous Organelles

Locations of Protein Synthesis ER Associated Ribosomes proteins translocate co-translationally into ER lumen subsequent processing & transport through Golgi for secretory vesicles and lysosomes Cytoplasmic Ribosomes proteins translocated post-translationally into membranous organelles (except Golgi, ER and lysosome)

Routes of Protein Trafficking Copyright 2008 by Saunders/Elsevier. All rights reserved.

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Routes of Intracellular Protein Movement Cytosolic (free) ribosomes Cytosolic proteins Imported into organelles Mitochondria, Chloroplasts, Peroxisomes, Nucleus Anchored facing cytoplasmic face of membranes C-tail anchored proteins in mitochondrial outer membrane and ER membrane Lipid anchored proteins on inner face of plasma membrane (via ER) ER-associated ribosomes Endomembrane system proteins ER, Golgi, lysosome, vesicles, plasma membrane, secreted proteins

Targeting signals that direct proteins synthesized in cytoplasm to organelles Fig. 18-1 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Translocons used by polypeptides to cross membranes in eukaryotes Fig. 18-2 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Directing Traffic Signal sequences Signal Recognition Particles Mostly N-terminal, C-terminal Occasionally internal Often cleaved following transport event Signal Recognition Particles Bind signal sequences Chaperone proteins Hsp Translocons Protein complexes to move proteins through/into membranes

Trafficking of Cytosolically Synthesized Proteins Import into organelles (transmembrane and lumenal) Mitochondrial import Chloroplast import Peroxisome import Translocation across plasma membrane Insertion in cytoplasmic face of plasma membrane

Mitochondrial Protein Import Four Destinations Outer membrane Innermembranous space Inner membrane Matrix TOMs Translocase of the outer mitochondrial membrane TIMs Translocase of the inner mitochondrial membrane Tiny TIMs escorts of the innermembranous space Signal Presequences 10-70 a.a. Basic, hydroxylate, & hydrophobic

Import of proteins into the mitochondrial matrix TOM & TIM Function Import of proteins into the mitochondrial matrix Fig. 18-4 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Facilitate Import into the Inner Membrane Tiny TIMs and TIM22 Facilitate Import into the Inner Membrane Fig. 18-5 Copyright 2008 by Saunders/Elsevier. All rights reserved.

TOC & TIC Import of proteins into the chloroplast thylakoid

Peroxisome Import Signal sequences PTS1 Recognized by PEX5 -SKL-COOH - RLxxxxxH/QL---

Peroxisome Formation

Translocation of ER-Associated Ribosome Synthesized Proteins

Endoplasmic reticulum (ER) Fig. 20-1 Hypothesis for the evolution of the ER Fluorescence micrograph of a cell with ER marked with a GFP fusion protein Copyright 2008 by Saunders/Elsevier. All rights reserved.

Domains of the endoplasmic reticulum Copyright 2008 by Saunders/Elsevier. All rights reserved. Fig. 20-2

Docking of Ribosomes to ER

Signal Peptide or Signal Sequence typically 15-35 residues shorter than those on cytosolically synthesized proteins Hydrophobic core of ~6 residues Examples MKLSLVAAMLLLLSAARA BiP MHYCVLSAFLILHLVTVAL TGF-b2 MRPSGTAGAALLALLAALCPRA EGFR

Signal Recognition Particle Ribonucleoprotein complex translocation Ribosome binding Signal peptide binding

Sec61 ER translocon

Structure of Sec61 translocon Fig. 20-6 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Targeting proteins to the ER lumen & membrane Fig. 20-7 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Targeting proteins to the ER membrane Fig. 20-7 Copyright 2008 by Saunders/Elsevier. All rights reserved.

Translocation of Various Classes of ER Proteins lumenal proteins Type 1 transmembrane proteins Type 2 transmembrane proteins multipass transmembrane proteins

Post-Translational Events II ER & Golgi Processes

Rough Endoplasmic Reticulum

Glycolsylation O-linked – serine, threonine, hydroxylysine (collagen) N-linked - asparagine The sugars GalNac – N-acetylgalactosamine GlcNac – N-acetylglucosamine NANA – N-acetylneuraminic acid (sialic acid) Gal – galactose Glc – glucose Man – mannose Fuc – fucose Dolicholpyrophosphoryl oligosaccharide All sugars are linked to nucleoside mono- or diphosphate UDP-gal, UDP-GalNac, UDP-GlcNac GDP-man CMP-NANA Dolichol pyrophosphate

Hydroxylated amino acids

Glyco-nucleoside Phosphates Dolichol phosphate CMP-N-acetylneuraminic acid CMP-sialic acid UDP-galactose UDP-N-acetylglucosamine GDP-mannose

O-linked Glycosylation Small chains of sugars N-acetylgalactosamine (GalNac) is first sugar on S orT GalNac transferase in ER and cis-golgi Additional sugars added in cis, medial & trans-golgi Gal added by galactosyl transferase NANA added last Each sugar addition is catalyzed by specific enzyme

N-linked Glycosylation Initial step in ER Dolichol pyrophosphoryl oligosaccharide 14 sugar moiety attached to dolichol pyrophosphate (GalNac)2Man9Glc3 Dolichol phosphate in ER membrane Sugars added by cytostolic glycosyltransferases Moiety flips in ER membrane to face lumen Transferred to asparagine by oligosaccharyl transferase (oligosaccharide-protein transferase) Target sequence N-X-S and N-X-T (X = P) Subsequent remodeling of sugar moiety in ER & golgi N-linked chains very long and extensively branched

O- & N-linked Oligosaccharide Chains

N-linked Glycolsylation

GPI – Linkages GPI – linked proteins are attached to GPI in ER GPI in ER membrane C-term of protein attached to sugar residue on phosphatidyl inositol GPI linked protein will be attached to outer leaflet of plasma membrane

Protein Folding Occurs in ER Lumen

Misfolded Proteins Reverse Translocate to Proteasome EDEM

Unique Signaling from ER to Make More ER Pathway activated by stresses that result in more unfolded proteins heat unfavorable pHs

Unfolded protein responses in the ER lumen Fig. 20-11 Activating transcription factor 6 Inositol requiring 1 PKR-like ER-kinase/pancreatic eIF2a kinase Copyright 2008 by Saunders/Elsevier. All rights reserved.

Sterol & Lipid Biosynthesis Phospholgycerides in ER Sphingolipids in Golgi Lipid profile asymmetry established via phospholipid exchange proteins and selective vesicular transport due to association between carrier proteins and certain lipid types

Regulation of HMG-CoA Reductase

On to the Golgi http://youtu.be/rvfvRgk0MfA http://youtu.be/u2lieHDDYPY http://youtu.be/rioXHf97cvs