1. Set up 110 µl mix for each primer/DNA combo on ice!1. 1.1 µl 100x F primer (1 pMol/µl = 1µM final [])2. 1.1 µl 100x R primer3. 11 µl 10x PCR buffer4. 2.2 µl 10 mM dNTP (200 µM final [])5. 2 µl DNA 6. 91.9 µl water7. 0.66 µl Taq polymerase

2. Split into 5 tubes @ 20 µl/tube3. Run on 50- 65 T gradient

PROTEIN TARGETINGAll proteins are made with an “address” which determines their final cellular location

Addresses are motifs within proteins

E. coli protein targeting1.Tat: for periplasmic redox proteins & thylakoid lumen!2.Sec pathway3.Periplasmic proteins with the correct signals (exposed after cleaving signal peptide) are exported by XcpQ system

2 Protein Targeting pathwaysProtein synthesis always begins on free ribosomes In cytoplasm1) Post -translational: proteins of plastids, mitochondria, peroxisomes and nuclei 2) Endomembrane system proteins are imported co-translationally

SIGNAL HYPOTHESIS• Protein synthesis begins on free ribosomes in cytoplasm• Endomembrane proteins have "signal sequence"that directs them to RER• SRP (Signal Recognition Peptide) binds signal sequence when it pops out of ribosome & swaps GDP for GTP

SIGNAL HYPOTHESISSRP stops protein synthesis until it binds “docking protein”(SRP receptor) in RER Ribosome binds Translocon & secretes protein through it as it is madeBiP (a chaperone) helps the protein fold in the lumensecretion must be cotranslational

Subsequent eventsSimplest case:

1) signal is cleaved within lumen by signal peptidase2) BiP helps protein fold correctly3) protein is soluble inside lumen

proteins embedded in membranesprotein has a stop-transfer sequence

too hydrophobic to enter lumentherefore gets stuck in membraneribosome releases translocon, finishes job in cytoplasm

More ComplicationsSome proteins have multiple trans-membrane domains (e.g. G-protein-linked receptors)

More ComplicationsExplanation: combinations of stop-transfer and internal signals-> results in weaving the protein into the membrane

Sorting proteins made on RERSimplest case: no sorting• proteins in RER lumen are secreted

Sorting proteins made on RERSimplest case: no sorting• proteins in RER lumen are secreted• embedded proteins go to plasma membrane

Sorting proteins made on RER

Redirection requires extra information:

Sorting proteins made on RER

Redirection requires extra information:

1) specific motif

2) receptors

Sorting proteins made on RER ER lumen proteins have KDEL (Lys-Asp-Glu-Leu) motif Receptor in Golgi binds & returns these proteinsER membrane proteinshave KKXX motif

Sorting proteins made on RERGolgi membrane proteins • cis- or medial- golgi proteins are marked by sequences in the membrane-spanning domain

• trans-golgi proteins have a tyrosine-rich sequence in their cytoplasmic C-terminus

Sorting proteins made on RERPlant vacuolar proteins are zymogens (proenzymes)

signal

signal

VTS

VTS

Barley aleurain

Barley lectin

mature protein

mature protein

Sorting proteins made on RERPlant vacuolar proteins are zymogens (proenzymes), cleaved to mature form on arrival• targeting motif may beat either end of protein

signal

signal

VTS

VTS

Barley aleurain

Barley lectin

mature protein

mature protein

Sorting proteins made on RERlysosomal proteins are targeted by mannose 6-phosphate M 6-P receptors in trans-Golgi direct protein to lysosomes (via endosomes)M 6-P is added in Golgi by enzyme that recognizes lysosomal motif