Intracellular Compartments and Protein Sorting

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Transcript Intracellular Compartments and Protein Sorting

Organization Of Cell
• Contains membraneenclosed organelles
• Nucleus
• Cytoplasm
– Cytoplasmic organelles
– Cytosol
Transporting Proteins To
• 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
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
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
• 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
• Oligosaccharide to be
transferred attached to
Synthesis Of Dolichol-linked
• Stepwise addition of sugar
• Nucleotide-sugar
intermediates donate sugars
• Monosaccharide-linked
dolichol molecules transfer
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
• 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
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
Golgi Stack
cis cisterna
medial cisterna
trans cisterna
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
• Two classes formed by
modifications to precursor in Golgi
• 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
• Constitutive secretory pathway: transport
continually from TGN to plasma membrane
• Regulated secretory pathway: store in
secretory vesicles until stimulated
• 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
– Phagocytosis
• Autophagy
Receptor-mediated Endocytosis
• 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
Sorting In Early Endosome
• Endocytoic vesicles fuse
with early endosomes
• Ligand-receptor dissociation
• Possible fates of receptor:
recycling, transcytosis,
Endocytic Pathway Of LDL
• LDL receptor recycled to plasma membrane
• LDL degraded in lysosome to release free cholesterol
From Early Endosomes To
• Early endosomes form
multivesicular bodies by
enclosing invaginations
• Turn into late endosomes that are
more acidic
• Form lysosomes by receiving
hydrolases, further acidification