Download Transport of Cytoplasmically Synthesized Proteins into Membranous

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
– 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
TOM & TIM Function
Import of proteins into the mitochondrial matrix
Fig. 18-4
Copyright 2008 by Saunders/Elsevier. All rights reserved.
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
• PTS2
• - RLxxxxxH/QL---
Peroxisome Formation
Translocation of ER-Associated
Ribosome Synthesized Proteins
Endoplasmic reticulum (ER)
Fig. 20-1
Hypothesis for the evolution of the ER
Copyright 2008 by Saunders/Elsevier. All rights reserved.
Fluorescence micrograph
of a cell with ER marked
with a GFP fusion protein
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
– MHYCVLSAFLILHLVTVAL
– MRPSGTAGAALLALLAALCPRA
BiP
TGF-b2
EGFR
Signal Recognition Particle
• Ribonucleoprotein complex
Ribosome binding
translocation
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
Copyright 2008 by Saunders/Elsevier. All rights reserved.
Fig. 20-7
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)
O- & N-linked
Oligosacchari
de 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
• Pathway activated by stresses
thatER
result in more
unfolded proteins
–heat
–unfavorable
pHs
Unfolded protein responses in the ER lumen
Activating transcription factor 6
Fig. 20-11
Inositol requiring 1
PKR-like ERkinase/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
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