Organization Of Cell

• Contains membrane-enclosed 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

• 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

• 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

– Acts to stop transfer– Remains as membrane-spanning segment

• N-terminal sequence for transport and start transfer• Additional internal hydrophobic 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

• Transfer of preformed oligosaccharide precursor

• Catalyzed by oligosaccharyl transferase

• Oligosaccharide to be transferred attached to dolichol

Glycosylation In ER

• Stepwise addition of sugar resides

• Nucleotide-sugar intermediates donate sugars

• Monosaccharide-linked dolichol molecules transfer sugars

Synthesis Of Dolichol-linked Oligosaccharide

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

• Some proteins destined for plasma membrane• Hydrophobic C-terminal sequence• C-terminus cut and preassembled GPI attached

Addition Of GPI Anchor

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

• introduces curvature leading to formation of bud• linked to transmembrane cargo receptors by adaptins• removed after transport vesicle is pinched off

Clathrin coat:

Formation Of Clathrin-coated Vesicle

CGNGolgi Stack

cis cisternamedial cisternatrans cisterna

TGN

ER → CGN → cis-, medial-, trans cisternae → TGN

Organization Of Golgi Apparatus

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

• Sorting signal for retrieval of ER proteins that enter GolgiMembrane proteins: KKXX- (COO-)Soluble proteins: KDEL- (COO-)

• Transmembrane receptor for KDEL that binds coat proteins

Golgi → ER

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

– defective hexosaminidase A gene– accumulation of ganglioside GM2

• I-cell disease– most hydrolases missing from lysosomes– inclusion bodies– defective GlcNAc phosphotransferase gene

• Hurler’s disease– defective -L-iduronase gene

• Gaucher disease – defective glucocerebrosidase gene

• Tay-Sachs disease

Protein Sorting In TGN

• Lysosomes• Constitutive secretory pathway

– Transport vesicles from TGN to plasma membrane– Default pathway

– Sorting signal targets to special secretory vesicles• Regulated secretory pathway

• 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

• Endocytosis– Pinocytosis– Receptor-mediated

endocytosis– Phagocytosis

• Autophagy

Delivering materials to lysosomes for digestion:

Endocytic/Degradation Pathways

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

• Early endosomes form multivesicular bodies by enclosing invaginations

• Turn into late endosomes that are more acidic

• Form lysosomes by receiving hydrolases, further acidification

From Early Endosomes To Lysosomes