Gene expression

Gene expression

• Gene expression

• Transcription
• Translation

• Functional proteins: post-translational modifications

• Phosphorylation
• Glycosylation

• Sorting: targeting to appropriate organelles

• Modulation by extracellular signals

• Covalent modification
• Association with other molecules

• Degradation

Sorting and Secretory pathways

• diverged in trans-Golgi

• Constitutive pathway
• many soluble proteins
• Regulated pathway
• stored in secretory vesicles
• active transport from cytosol to vesicles
• complexed with macromolecules (e.g. proteglycans)

Regulated secretory pathway

• In response to extracellular stimuli: hormone, transmitters, digestive enzymes

• stored in secretory vesicles

• active transport from cytosol to vesicles

• complexed with macromolecules (e.g. proteglycans) to reach high concentration

Three pathways in Golgi

• to lysosome

• with mannose-6-phosphate, via late endosome to lysosome

• to secretory pathways

• Constitutive: to apical or basolateral domain
• Regulated

“Roadmap” for traffic

• Transport

• Gated transport: cytosol and nucleus via nuclear pore complexes
• Membrane transport: via membrane-bound translocators; unfolded
• Vesicle transport: vesicles

• Sorting signals

Membrane transport

• Membrane-bound translocators

• Unfolding of protein to be transported

• Passing through a topologically distinct space: cytosol to ER; cytosol to mitochondria

Equivalent space for transport

• Cycles of budding and fusion permits transport of molecules

Sorting signals

• Signal sequences

• 15-60 aa
• Removed by signal peptidase when reaching the target

• Signal patch

• Usually non-continuous stretch of sequences
• Exposed when appropriately folded

Sorting signals

Signal sequences

• Function: specify the direction for destination

• for initial transfer to the ER: with a signal sequence at N-terminus; consisting of 5-10 hydrophobic aa

• Go forward Golgi: most proteins
• Return to ER (ER residents): with a specific sequence of 4 aa at C-terminus
• Go to mitochondria: positively charged amino acids alternate with hydrophobic ones
• Go to peroxisome: with a signal peptide of 3 characteristic at C terminus

Signal sequences

Experiments to demonstrate “zip code”

• Swap the signal sequence:

• e.g.: adding the ER signal sequences to a cytosolic protein results in the retention of this protein in ER

• Hyprophobicilty: maybe a determing factor of signal sequences

Günter Blobel

Effect of misdirected protein

Overview of protein sorting

Protein sorting

• ER

• ER-Golgi intermediate

• Glogi network

Glycosylation in ER

Lysosomal protein: phosphorylation of mannose

Sorting in Golgi

• Export

• Lysosome
• Secretion
• Constitute
• Regulated

• Retain in Golgi

• transmembrane

Transport to plasma membrane

• TGN

• Apical domain

• Basolateral domain

Endocytosis

• Phagocytosis (cell eating)

• Ingestion of large particles (e.g. bacteria)
• In specialized cells

• Pinocytosis (cell drinking)

• Up-take of fluids or macromolecules in small vesicles
• Receptor-mediated endocytosis
• Common among eukaryotic cells

Phagocytosis

• Pseudopodia

• Phagosome

• Phagolysosome

Receptor-mediated endocytosis

• Clathrin-coated pits

• Clathrin-coated vesicles

• Example: cholesterol, LDL, LDL receptor

LDL: low density lipoprotein

• Core

• ~1500 Cholesterol ester

• Coat

• ~500 cholesterol
• ~ 800 phospholipid
• 1 Apoprotein B100

LDL receptor

• MS Brown, JL Goldstein

• Familial hypercholesterolemia

• LDL-binding domain

• Internalization signal

LDL receptor

• Internalization signal: Tyr

Clathrin-coated pits

Lysosomal proteins in clathrin-coated pits

Sorting in early endosome

• Acidic ph by H+ pump for dissociation of proteins

Recycling of synaptic vesicles

Protein sorting by transcytosis

• Membrane protein in apical domain

• Secretory proteins from bloodstream

Lysosomal system

• For membrane-bound proteins and proteins taken by endocytosis

• Multiple acid proteases (cathepsins) and other hydrolases

• Studied with weak bases to inhibit lysosomal acidification or lysosomal inhibitors (E64)