CELL BIOLOGY FINAL
EXAM REVIEW
2pm: Tuesday April 18th, 2023
,Targeting and Sorting Proteins to their Locations of Function Requires Signal Sequences
• Transcription (DNA → RNA) occurs in the nucleus
• RNA is exported from the nucleus to the cytoplasm
• Translation begins on a free-floating ribosome in the cytoplasm
• Cytoplasmic proteins are fully translated and remain / function in the cytosol
• Proteins that do not function in cytosol are targeted to an organelle by a signal sequence:
o Mitochondrial proteins: completely translated in cytosol; contain mitochondrial
matrix targeting sequence of ~20-50 amphipathic (hydrophilic + hydrophobic)
amino acids near N-terminus
▪ Two TOMs (translocon of outer mitochondrial membrane) act as receptor
then translocon—move protein into intermembrane space
▪ TIM (Translocon of inner mitochondrial membrane) allows protein into
mitochondrial matrix; pulled through by HSP70
▪ Matrix processing protease cleaves off target sequence
o Nuclear proteins: contain nuclear localization sequence (NLS)
i) Importin recognizes NLS and brings protein from cytosol into
nucleus via nuclear pore complex (NPC)
ii) GEF energizes Ran-GDP → Ran-GTP (high affinity for
importin)
iii) Importin binds Ran-GTP and releases cargo protein
iv) GAP hydrolyzes Ran-GTP → Ran-GDP, resetting and allowing
importin to dissociate and go back into cytosol
o Secretory pathway proteins (ER): contain an N-terminal signal sequence
▪ Transported to the ER via Co-Translational Translocation
i) First ~20 aa translated in cytosol = signal sequence
ii) Recognizes by signal recognition particle (SRP)
iii) Translation stops and SRP brings ribosome/peptide
complex to RER
iv) SRP receptor on ER membrane binds SRP (must both be
GTP bound to bind; hydrolyze to dissociate SRP)
v) SRP receptor binds Translocon
vi) Translocon opens, puts N-terminus of peptide in ER lumen
vii) Signal sequence cleaved off by signal peptidase
viii) Peptide moved into lumen
ix) Translation resumes/completed in ER lumen
• Once in the ER, proteins may be sorted to:
o Anterograde Transport: ER → cis Golgi
o Cis Golgi → trans Golgi
▪ From trans Golgi:
• Retrograde Transport: trans Golgi → ER
• Trans Golgi → Late Endosome → Lysosome
• Trans Golgi → Plasma Membrane
1
,Anterograde Transport: ER → cis Golgi
• Di-acidic Sorting signal (DXE)
o On cytosolic domain of ER transmembrane protein
i) GTP-binding proteins promote association of COPII coat proteins at ER membrane
ii) COPII coat proteins recognize DXE signal
iii) Cause budding of ER membrane to form COPII-coated vesicle
iv) Vesicle moves ER → cis Golgi
v) GTP hydrolysis to release coat proteins
vi) Release of coat proteins exposes SNARE proteins
vii) Snares on vesicle and Golgi membrane twist around one another until membranes fuse
viii) Contents of vesicle released into Golgi lumen
ix) ATP hydrolysis to release SNARE proteins
• Cystic Fibrosis: ∆F508 deletion in CFTR; DXE not exposed to cytosol; cannot bind
COPII coat proteins—cannot be transported to plasma membrane to function, remains
stuck in ER
Retrograde Transport: Golgi → ER
• KDEL sequence
o Found on ER proteins, allow transport back to ER if accidentally go to Golgi
i) KDEL sequence exposed to Golgi’s lower pH than ER, undergoes conformational change
which allows it to bind KDEL receptor in Golgi
ii) Binding causes conformational change in KDEL receptor, which exposes its C-terminal
KKXX sequence (di-basic sorting signal) to the cytosol
iii) KKXX recognized by COPI coat proteins
iv) Cause budding of Golgi membrane to form COPI-coated vesicle
v) Vesicle moves Golgi → ER
vi) GTP hydrolysis to release coat proteins
vii) Release of coat proteins exposes SNARE proteins
viii) Snares on vesicle and ER membrane twist around one another until membranes fuse
ix) Contents of vesicle released into ER lumen
x) ATP hydrolysis to release SNARE proteins
• Chaperone proteins in ER (BiP, calreticulin, calnexin) have KDEL
Trans Golgi → Late Endosome → Lysosome
• Mannosse-6-Phosphate (M6P) signal
o Added to lysosomal proteins in Golgi
▪ GlcNAc phosphate transferase adds phosphate to Mannose residue of
oligosaccharide (sugar added in ER)
▪ Phosphodiesterase cleaves phosphodiester to release protein from enzyme
i) At pH ~6.5 in Golgi, M6P signal sequence recognized by M6P receptor
ii) Recruits AP1 (adaptor protein) and Clathrin proteins
2
, iii) Causes budding of Golgi membrane to form Clathrin-coated vesicle
iv) Coat proteins shed
v) Vesicle moves Golgi → late endosome
vi) pH ~5-5.5 inn late endosome promotes release of M6P-tagged protein from M6P receptor
vii) Late endosome fuses with lysosome, releasing lysosomal protein into lysosome
• Inclusion cell (I-cell) Lysosomal Storage disease
o No functional GlcNAc phosphotransferase enzyme
▪ M6P signal not added to lysosomal proteins
• End up secreted out of cell by default
o Lysosome doesn’t break down waste
▪ Developmental / neurological abnormalities, fatal in early childhood
Trans Golgi → Plasma Membrane (Membrane or Secreted Proteins)
• Default
Receptor-Mediated Endocytosis: Selective Internalization of Extracellular Molecules
Endocytosis of LDL
• Low-density Lipoprotein (LDL): large water-soluble particle which mediates cholesterol
transport
• LDL Receptor:
o C-terminal NPXY sorting signal
o Transmembrane domain
o N-terminal extracellular domain
▪ Ligand-binding arm (-)
▪ Β-propeller domain (His residues can be protonated in acidic conditions)
i) LDL particle recognized by LDL receptor within Clathrin-coated pits in plasma
membrane
ii) At extracellular pH 7, ligand-biding arm of LDL receptor binds Apolipoprotein B (belt
around LDL)
iii) Conformational change in LDL receptor which exposes NPXY sorting signal into
cytosol (inside cell)
iv) Recruits AP2 coat proteins and Clathrin
v) Clathrin-coated endocytic vesicle forms by budding off membrane using Dynamin
(squeezes around neck of bud) and GTP hydrolysis
vi) Vesicle moves PM → late endosome
vii) pH ~5-5.5 in late endosome lumen causes LDL receptor to release LDL particle
o Acidic pH causes protonation of Histidine residues in β-propeller domain of LDL
receptor
o (+) β-propeller domain binds (-) ligand-binding arm
▪ Causes ligand binding arm to release LDL particle
viii) Late endosome fuses with lysosome
ix) Lysosome breaks down LDL particles
3
EXAM REVIEW
2pm: Tuesday April 18th, 2023
,Targeting and Sorting Proteins to their Locations of Function Requires Signal Sequences
• Transcription (DNA → RNA) occurs in the nucleus
• RNA is exported from the nucleus to the cytoplasm
• Translation begins on a free-floating ribosome in the cytoplasm
• Cytoplasmic proteins are fully translated and remain / function in the cytosol
• Proteins that do not function in cytosol are targeted to an organelle by a signal sequence:
o Mitochondrial proteins: completely translated in cytosol; contain mitochondrial
matrix targeting sequence of ~20-50 amphipathic (hydrophilic + hydrophobic)
amino acids near N-terminus
▪ Two TOMs (translocon of outer mitochondrial membrane) act as receptor
then translocon—move protein into intermembrane space
▪ TIM (Translocon of inner mitochondrial membrane) allows protein into
mitochondrial matrix; pulled through by HSP70
▪ Matrix processing protease cleaves off target sequence
o Nuclear proteins: contain nuclear localization sequence (NLS)
i) Importin recognizes NLS and brings protein from cytosol into
nucleus via nuclear pore complex (NPC)
ii) GEF energizes Ran-GDP → Ran-GTP (high affinity for
importin)
iii) Importin binds Ran-GTP and releases cargo protein
iv) GAP hydrolyzes Ran-GTP → Ran-GDP, resetting and allowing
importin to dissociate and go back into cytosol
o Secretory pathway proteins (ER): contain an N-terminal signal sequence
▪ Transported to the ER via Co-Translational Translocation
i) First ~20 aa translated in cytosol = signal sequence
ii) Recognizes by signal recognition particle (SRP)
iii) Translation stops and SRP brings ribosome/peptide
complex to RER
iv) SRP receptor on ER membrane binds SRP (must both be
GTP bound to bind; hydrolyze to dissociate SRP)
v) SRP receptor binds Translocon
vi) Translocon opens, puts N-terminus of peptide in ER lumen
vii) Signal sequence cleaved off by signal peptidase
viii) Peptide moved into lumen
ix) Translation resumes/completed in ER lumen
• Once in the ER, proteins may be sorted to:
o Anterograde Transport: ER → cis Golgi
o Cis Golgi → trans Golgi
▪ From trans Golgi:
• Retrograde Transport: trans Golgi → ER
• Trans Golgi → Late Endosome → Lysosome
• Trans Golgi → Plasma Membrane
1
,Anterograde Transport: ER → cis Golgi
• Di-acidic Sorting signal (DXE)
o On cytosolic domain of ER transmembrane protein
i) GTP-binding proteins promote association of COPII coat proteins at ER membrane
ii) COPII coat proteins recognize DXE signal
iii) Cause budding of ER membrane to form COPII-coated vesicle
iv) Vesicle moves ER → cis Golgi
v) GTP hydrolysis to release coat proteins
vi) Release of coat proteins exposes SNARE proteins
vii) Snares on vesicle and Golgi membrane twist around one another until membranes fuse
viii) Contents of vesicle released into Golgi lumen
ix) ATP hydrolysis to release SNARE proteins
• Cystic Fibrosis: ∆F508 deletion in CFTR; DXE not exposed to cytosol; cannot bind
COPII coat proteins—cannot be transported to plasma membrane to function, remains
stuck in ER
Retrograde Transport: Golgi → ER
• KDEL sequence
o Found on ER proteins, allow transport back to ER if accidentally go to Golgi
i) KDEL sequence exposed to Golgi’s lower pH than ER, undergoes conformational change
which allows it to bind KDEL receptor in Golgi
ii) Binding causes conformational change in KDEL receptor, which exposes its C-terminal
KKXX sequence (di-basic sorting signal) to the cytosol
iii) KKXX recognized by COPI coat proteins
iv) Cause budding of Golgi membrane to form COPI-coated vesicle
v) Vesicle moves Golgi → ER
vi) GTP hydrolysis to release coat proteins
vii) Release of coat proteins exposes SNARE proteins
viii) Snares on vesicle and ER membrane twist around one another until membranes fuse
ix) Contents of vesicle released into ER lumen
x) ATP hydrolysis to release SNARE proteins
• Chaperone proteins in ER (BiP, calreticulin, calnexin) have KDEL
Trans Golgi → Late Endosome → Lysosome
• Mannosse-6-Phosphate (M6P) signal
o Added to lysosomal proteins in Golgi
▪ GlcNAc phosphate transferase adds phosphate to Mannose residue of
oligosaccharide (sugar added in ER)
▪ Phosphodiesterase cleaves phosphodiester to release protein from enzyme
i) At pH ~6.5 in Golgi, M6P signal sequence recognized by M6P receptor
ii) Recruits AP1 (adaptor protein) and Clathrin proteins
2
, iii) Causes budding of Golgi membrane to form Clathrin-coated vesicle
iv) Coat proteins shed
v) Vesicle moves Golgi → late endosome
vi) pH ~5-5.5 inn late endosome promotes release of M6P-tagged protein from M6P receptor
vii) Late endosome fuses with lysosome, releasing lysosomal protein into lysosome
• Inclusion cell (I-cell) Lysosomal Storage disease
o No functional GlcNAc phosphotransferase enzyme
▪ M6P signal not added to lysosomal proteins
• End up secreted out of cell by default
o Lysosome doesn’t break down waste
▪ Developmental / neurological abnormalities, fatal in early childhood
Trans Golgi → Plasma Membrane (Membrane or Secreted Proteins)
• Default
Receptor-Mediated Endocytosis: Selective Internalization of Extracellular Molecules
Endocytosis of LDL
• Low-density Lipoprotein (LDL): large water-soluble particle which mediates cholesterol
transport
• LDL Receptor:
o C-terminal NPXY sorting signal
o Transmembrane domain
o N-terminal extracellular domain
▪ Ligand-binding arm (-)
▪ Β-propeller domain (His residues can be protonated in acidic conditions)
i) LDL particle recognized by LDL receptor within Clathrin-coated pits in plasma
membrane
ii) At extracellular pH 7, ligand-biding arm of LDL receptor binds Apolipoprotein B (belt
around LDL)
iii) Conformational change in LDL receptor which exposes NPXY sorting signal into
cytosol (inside cell)
iv) Recruits AP2 coat proteins and Clathrin
v) Clathrin-coated endocytic vesicle forms by budding off membrane using Dynamin
(squeezes around neck of bud) and GTP hydrolysis
vi) Vesicle moves PM → late endosome
vii) pH ~5-5.5 in late endosome lumen causes LDL receptor to release LDL particle
o Acidic pH causes protonation of Histidine residues in β-propeller domain of LDL
receptor
o (+) β-propeller domain binds (-) ligand-binding arm
▪ Causes ligand binding arm to release LDL particle
viii) Late endosome fuses with lysosome
ix) Lysosome breaks down LDL particles
3
