Download Intracellular Compartments and Protein Sorting

Organization Of Cell
• Contains membraneenclosed organelles
• Nucleus
• Cytoplasm
– Cytoplasmic organelles
– Cytosol
Transporting Proteins To
Organelles
• Synthesis begins in cytosol
• Several mechanisms for
transporting to organelles
Sorting Signals
• Segment(s) of amino acids direct protein to an organelle
• Recognized by sorting receptors
• Proteins with no sorting signal remain in cytosol
Examples Of Signal Sequences
• Sequence variability; physical properties often important
Studying Signal Sequences
• Functional signal sequences determined by experimental
manipulation of proteins to alter their localization
Nuclear Pore Complex
• Multi-protein complex composed of nucleoporins
• Diffusion of small molecules
• Selective gate for proteins
Nuclear Import & Export
• Nuclear import receptor binds NLS of protein to be imported
• Cargo-bound import receptor binds nucleoporins
• Nuclear export is similar: export receptor binds to NES
Functions Of Peroxisomes
• Enzymes produce and consume H202 to oxidize organic substrates
RH2 + O2 → R + H202 (various enzymes)
H202 + R'H2 → R' + 2H20 (catalase)
2 H202 → 2H20 + O2 (catalase)
• Synthesis of plasmalogens
Import Into Peroxisomes
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Signal sequence often at C-terminus
Some proteins with sequence near N-terminus
Peroxins (receptors, docking proteins) participate in transport
Inherited defects in peroxin genes such as Zellweger syndrome
Transport Into Mitochondria
• Have own genome for some proteins;
maternally inherited
• Nuclear genome encodes most proteins;
synthesized in cytosol and imported
Endoplasmic Reticulum
• Site of synthesis for all proteins destined for
secretion, the plasma membrane, lysosomes,
endosomes, the Golgi, or the ER itself
Docking Protein Onto ER
Membrane
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Signal sequence contains hydrophobic amino acids
SRP binds to signal sequence as it emerges from ribosome
Co-translational transport onto ER membrane
Start transfer through translocator as translation continues
Soluble Protein Into ER Lumen
• Signal sequence at N-terminus
• Co-translational transport and translocation through membrane
• Cleavage of signal sequence
ER Transmembrane Protein With
N-Terminal Signal
• N-terminal sequence for transport and start transfer
• Additional internal hydrophobic segment
– Acts to stop transfer
– Remains as membrane-spanning segment
ER Transmembrane Protein With
Internal Signal
• Internal sequence for transport and start-transfer
• Remains as membrane-spanning segment
• Two orientations of signal sequence
ER Multi-pass Transmembrane
Protein
• Multiple internal start and stop tranfer sequences
N-Linked Glycosylation
• Glycoproteins made in ER
• Oligosaccharide precursor added
to asparagine residues in ER
• Processing in Golgi removes
some sugar residues
Glycosylation In ER
• Transfer of preformed
oligosaccharide precursor
• Catalyzed by oligosaccharyl
transferase
• Oligosaccharide to be
transferred attached to
dolichol
Synthesis Of Dolichol-linked
Oligosaccharide
• Stepwise addition of sugar
resides
• Nucleotide-sugar
intermediates donate sugars
• Monosaccharide-linked
dolichol molecules transfer
sugars
O-Linked Glycosylation
• Oligosaccharide linked to hydroxyl groups of
serine, threonine, or hydroxylysine residues
• Occurs in Golgi
Protein Folding In ER
• Chaperones aid in folding
• Improperly folded proteins enter cytosol through
translocator; deglycosylated, ubquitylated, and degraded
Addition Of GPI Anchor
• Some proteins destined for plasma membrane
• Hydrophobic C-terminal sequence
• C-terminus cut and preassembled GPI attached
Vesicular Transport
• Vesicle buds off from one compartment and fuses with another
• Compartments that communicate are topologically equivalent
Protein Coats In Vesicular
Transport
• Cage of proteins covering cytosolic surface
• Concentrates membrane proteins and deforms membrane
Clathrin Structure
• Subunits associate into triskelion
• Convex framework of triskelions on cytosolic surface
Formation Of Clathrin-coated
Vesicle
Clathrin coat:
• introduces curvature leading to formation of bud
• linked to transmembrane cargo receptors by adaptins
• removed after transport vesicle is pinched off
Organization Of Golgi Apparatus
CGN
Golgi Stack
cis cisterna
medial cisterna
trans cisterna
TGN
ER → CGN → cis-, medial-, trans cisternae → TGN
Transporting From ER To CGN
• Exit signal on soluble
cargo interacts with
transmembrane receptor
• Exit signal on receptor
interacts with protein coat
ER Resident Proteins
Golgi → ER
• Sorting signal for retrieval of ER proteins that enter Golgi
Membrane proteins: KKXX- (COO-)
Soluble proteins: KDEL- (COO-)
• Transmembrane receptor for KDEL that binds coat proteins
Processing N-linked
Oligosaccharides
• Two classes formed by
modifications to precursor in Golgi
Complex
oligosaccharides
High-mannose
oligosaccharides
Lysosomes
• Controlled digestion
of macromolecules
Sorting By Recognizing M6P
• M6P added to lysosomal hydrolases in CGN
• Transmembrane M6P receptors in TGN
interact with coat proteins
Specific Addition Of M6P
• Signal patch recognized by GlcNAc phosphotransferase
Lysosomal Storage Diseases
• Genetic defects affecting lysosomal hydrolases
• Accumulation of undigested material in lysosomes
• Tay-Sachs disease
– defective hexosaminidase A gene
– accumulation of ganglioside GM2
• Gaucher disease
– defective glucocerebrosidase gene
• Hurler’s disease
– defective a-L-iduronase gene
• I-cell disease
– most hydrolases missing from lysosomes
– inclusion bodies
– defective GlcNAc phosphotransferase gene
Protein Sorting In TGN
• Lysosomes
• Constitutive secretory pathway
– Transport vesicles from TGN to plasma membrane
– Default pathway
• Regulated secretory pathway
– Sorting signal targets to special secretory vesicles
• Pathways initially involve ER signal sequence, SRP
Exocytosis
• Constitutive secretory pathway: transport
continually from TGN to plasma membrane
• Regulated secretory pathway: store in
secretory vesicles until stimulated
Endocytosis
• Material to be ingested becomes enclosed
by plasma membrane as it invaginates
• Buds off to form endocytic vesicles
Endocytic/Degradation Pathways
Delivering materials to
lysosomes for digestion:
• Endocytosis
– Pinocytosis
– Receptor-mediated
endocytosis
– Phagocytosis
• Autophagy
Receptor-mediated Endocytosis
Of LDL
• Cholesterol molecules in
LDL organized by protein
that binds to LDL receptor
• LDL receptor interacts
with clathrin-coated pit
• Mutation in LDL
receptor causes familial
hypercholesterolemia
Sorting In Early Endosome
• Endocytoic vesicles fuse
with early endosomes
• Ligand-receptor dissociation
• Possible fates of receptor:
recycling, transcytosis,
degradation
Endocytic Pathway Of LDL
• LDL receptor recycled to plasma membrane
• LDL degraded in lysosome to release free cholesterol
From Early Endosomes To
Lysosomes
• Early endosomes form
multivesicular bodies by
enclosing invaginations
• Turn into late endosomes that are
more acidic
• Form lysosomes by receiving
hydrolases, further acidification