Bridget, Jephte, Kristi, Matt, Teresa1. Set up 20 µl mix for each primer/DNA combo on ice!

1. 2 µl 10x F primer (1 pMol/µl = 1µM final [])2. 2 µl 10x R primer3. 1 µl DNA

2. We will prepare Phusion master mix for 130 µl total volume1. 26 µl 5x Phusion HF buffer2. 2.6 µl 10 mM dNTP (200 µM final [])3. 67.6 µl water4. 1.3 µl Phusion polymerase

3. Add 15 µl master mix to each rxn4. JKMT run at 58˚ C annealing T5. Bridget run on touchdown starting at 72˚ C annealing T

Testing internal primers1. Set up 20 µl mix for each primer/DNA combo on ice!

1. 2 µl 10x internal F primer (1 pMol/µl = 1µM final [])2. 2 µl 10x internal R primer3. 0.5 µl DNA = best PCR reaction

2. We will prepare master mix for 90 µl total volume1. 9 µl 10x Taq buffer2. 1.8 µl 10 mM dNTP (200 µM final [])3. 58.4 µl water4. 0.56 µl Taq polymerase

3. Add 15.5 µl master mix to each rxn4. Run at 50˚ C annealing T, 2 minutes/72˚ cycle

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

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

Glycosylation within ERAll endomembrane proteins are highly glycosylated on lumenal domains.Glycosylation starts in the ER, continues in the Golgi

Glycosylation within ERAll endomembrane proteins are highly glycosylated on lumenal domains.Glycosylation starts in ER, continues in Golgi

makes proteins more hydrophilicessential for proper functiontunicamycin poisons cellsGlycosylation mutants are even sicker

Glycosylation in RER1)(CH2O)n are assembled stepwise on dolichol-PO42) Transfer (CH2O)n to target asn

Glycosylation in RER1)(CH2O)n are assembled stepwise on dolichol-PO42) Transfer (CH2O)n to target asn3) remove 2 glucose& bind chaperoneIf good, remove gluc 3 & send to Golgi

Glycosylation in RERremove 2 glucose & bind to chaperoneIf good, remove gluc 3 & send to GolgiIf bad, GT adds glucose& try againEventually, send bad proteins to cytosol & eat them

Glycosylationnext modify (CH2O) n in Golgi

Remove some sugars & add others

Glycosylationnext modify (CH2O) n in Golgi

Remove some sugars & add othersdifferent rxns occur in different parts of Golgiwhy we separate Golgi into distinct regions

Post-translational protein targeting

Key features

1) imported after synthesis

Post-translational protein targeting

Key features1) imported after synthesis2) targeting information is motifs in protein a) which organelleb) site in organelle3) Receptors guide it to correct site4) no vesicles!

Protein targeting in Post-translational pathway

SKL (ser/lys/leu) at C terminus targets most peroxisomal matrix proteins = PTS1

In humans 3 are targeted by 9 aa at N terminus = PTS2

Defective PTS2 receptor causes Rhizomelic chondrodysplasia punctata

N CSKL

N CPTS2

Targeting peroxisomal proteins• Bind receptor in cytoplasm• Dock with peroxisomal receptors• Import protein w/ounfolding it!• Recycle receptors

Peroxisomal Membrane SynthesisMost peroxisomes arise by fissioncan arise de novo!Mechanism is poorly understood/ may involve ER!Only need PEX 3 & PEX 16 to import pex membrane prot

Protein import into nucleinuclear proteins are targeted by internal motifs

necessary & sufficient to target cytoplasmic proteins to nucleus

Protein import into nucleinuclear proteins are targeted by internal motifs

as in golgi, are not specific shapes cf sequencesReceptors bind objects of the right shape!

Protein import into nuclei

3 types of NLS (nuclear localization sequence)

1) basic residues in DNA-binding region

+ + + LZ

Protein import into nuclei3 types of NLS (nuclear localization sequence)

1) basic residues in DNA-binding region2) SV-40 KKKRK

KKKRK

+ + + LZ

Protein import into nuclei3 types of NLS (nuclear localization sequence)

1) basic residues in DNA-binding region2) SV-40 KKKRK3) bi-partite: 2-4 basic aa,10-20 aa spacer, 2-4 basic aa

KKKRK

+ + + LZ

+ +

+ +

Protein import into nuclei1) importin binds NLS importin binds complex2) escort to nuclear pores

•Pores decide who can enter/exit nucleus

Protein import into nuclei1) importin binds NLS, importin binds complex2) escort to nuclear pores 3) transporter changes shape, lets complex enter4) nuclear Ran-GTP dissociates complex 5) Ran-GTP returns importinto cytoplasm, becomes Ran-GDP. GTP -> GDP = nuclear import energy source6) Exportins return importin& other cytoplasmic prot

Protein import into cp and mito

Many common features

1) Pulse-chase experiments show most cp & mt proteins are made in cytoplasm as larger precursor (preprotein)

• both have N-terminal targeting peptide

• transit peptide in cp

• presequence in mito

•necessary & sufficient to target

Protein import into cp &mitoMany common features1) N-terminal transit peptide or presequence

• necessary & sufficient to target• usually removed upon arrival

Protein import into cp & mitoMany common features1) N-terminal transit peptide or presequence2) both need energy input

a) ATP for bothb) Mt also use Proton Motive Force (PMF)H+ gradient made by electron transportc) Cp also use GTP (but not PMF)

Protein import into cp & mito

1) N-terminal transit peptide or presequence

2) both need energy input

3) proteins unfold to enter, then refold inside

a) need chaperonins on both sides of membrane

i) chaperonins in cytosol unfold

ii) chaperonins inside refold

a) helps draw through membrane

Protein import into mitochondriaPrecursor has N-terminal targeting presequence

20 - 70 aa1. Many basic a.a (+ charge) = lys, arg2. Many hydroxylated a.a. (ser, thr)3. Segment can fold into -helix

mature proteinpresequencepresequence+ + +

Protein import into mitochondria

1) HSP70 binds & unfolds preprotein

Protein import into mitochondria1) HSP70 binds & unfolds preprotein2) Unfolded presequence binds MOM receptors (MOM19 & MOM72)

Protein import into mitochondria1) HSP70 binds & unfolds preprotein2) Unfolded presequence binds MOM receptors3) Unfolded protein is translocated through MOMcontroversy: do inner and outer membrane contact each other before protein import?

Protein import into mitochondria1) HSP70 binds & unfolds preprotein2) Unfolded presequence binds MOM receptors3) Unfolded protein is translocated through MOM4) Unfolded protein is translocated through MIMpresequence contacts MIM proteins

Protein import into mitochondria5) Chaperones in matrix refold protein2 different chaperones: mHSP70 & HSP60consumes ATP

Protein import into mitochondriaDriving forces for import:1) PMF (on +ve a.a.)2) Refolding (Brownian ratchet)3) ATP hydrolysis used to drive unfolding and refolding

Protein import into mitochondria6) Once protein is refolded, targeting sequence is removed

CP protein import1)chaperones in cytoplasm unfold preprotein2)transit peptide contacts receptor in OE

transit peptides:longer & less +ve than presequences Just a few changes convert transit peptide to presequence

CP protein import1) chaperones in cytoplasm unfold preprotein2) transit peptide contacts receptor in OE3) Unfolded protein is translocated through OE

• requires GTP•difference from mito

CP protein import1) chaperones in cytoplasm unfold preprotein2) transit peptide contacts receptor in OE3) Unfolded protein is translocated through OE4) Unfolded protein is translocated through IE

CP protein import5) Protein is folded on inside by chaperones6) transit peptide is removed

Energy for cp import1) GTP hydolysis:crossing OE2) Refolding (Brownian ratchet)3) ATP hydrolysis: un- & refolding

CP Protein importTargeting to other parts requires another motifHypothesis: proteins enter stroma first, thenfind their final destination

Proteomicsstudying all of the proteins present in a particular organism • Now that we have the genome, what do we do with it? • old way was to prepare 2-Dgels of proteins prepared fromthe cells being studied • first use isoelectric focusing to separate proteins by pI

Proteomicsold way was to prepare 2-D gels of proteins prepared from the

organisms or tissues being studied • first use isoelectric focusing to separate proteins by pI• Then use SDS-PAGE to separate by size

Proteomicsold way was to prepare 2-D gels of proteins prepared from the

organisms or tissues being studied • first use isoelectric focusing to separate proteins by pI• Then use SDS-PAGE to separate by size

ProteomicsUse 2D-SDS-PAGE to Separate by pI then size

Then ID each spot!• Sequencing

ProteomicsUse 2D-SDS-PAGE to Separate by pI then size Then ID each spot!

• Sequencing• Slow (1aa/hr)

ProteomicsUse 2D-SDS-PAGE to Separate by pI then size Then ID each spot!

• Sequencing• Slow (1aa/hr)• 98% accurate = 50 aalimit

MALDI (matrix assisted laser desorption ionization) to ionize peptides so they can be analyzed by mass spectrometry.

sample is dispersed in a large excess of matrix material which absorbs the incident laser

sample is dispersed in a large excess of matrix material which absorbs the incident laser

Short pulses of laser light focused on the sample cause the sample and matrix to volatilize

Short pulses of laser light focused on the sample cause the sample and matrix to volatilize

The ions formed are accelerated by high voltage & then drift down a flight tube where they separate according to mass

ProteomicsMALDI (matrix assisted laser desorption ionization) or ESI

(Electrospray ionization) to ionize peptides so they can be analyzed by mass spectrometry.

ProteomicsMALDI (matrix assisted laser desorption ionization) or ESI

(Electrospray ionization) to ionize peptides so they can be analyzed by mass spectrometry.

Identify by comparing peptide mass fingerprint with a library of theoretical mass spectra

ProteomicsMALDI (matrix assisted laser desorption ionization) or ESI

(Electrospray ionization) to ionize peptides so they can be analyzed by mass spectrometry.

Identify by comparing peptide mass fingerprint with a library of theoretical mass spectraOften do MS/MS: first collect all ions of a certain size, then refragment

ProteomicsThen ID each spot!

• Sequencing• MALDI (matrix assisted laser desorption ionization) or

ESI (Electrospray ionization) to ionize peptides so they can be analyzed by mass spectrometry.

• Identify by comparing the peptide mass fingerprint with a library of theoretical mass spectra

problems include • post-translational modifications that affect mass

ProteomicsUse 2-D gels to separate proteomeproblems include • post-translational modifications that affect mass • limits of detection

Also superimpose 2-D gels from different tissues or diseases to identify spots that increased or decreased in intensity

Many 2-D gel databases eg: www.expasy.ch/swiss-2dpage/-index.html

ProteomicsAlso superimpose 2-D gels from different tissues or diseases

to identify spots that increased or decreased in intensityMany 2-D gel databases eg: www.expasy.ch/swiss-2dpagesoftware such as Melanie (

http://us.expasy.org/melanie/) allows you to superimpose 2D gels to identify and quantitate spots, calculate mass and pI, measure differences between treatments, etc.

ProteomicsOther approaches1.Genome-wide 2–hybrid libraries

ProteomicsOther approaches1.Genome-wide 2–hybrid libraries2. multiple stage HPLC- MS

ProteomicsOther approaches1.Genome-wide 2–hybrid libraries2. multiple stage HPLC- MS3. Protein microarrays• antibodies

ProteomicsOther approaches1.Genome-wide 2–hybrid libraries2. multiple stage HPLC- MS3. Protein microarrays• Antibodies• N.A. probes

Proteomics3. Protein microarrays• Antibodies• N.A. probes• Specific proteins

ProteomicsProtein microarrays• Antibodies• DNA (or RNA) as probes• Specific proteins

Problems:1.proteins must retain their 3-D structure and function

ProteomicsProtein microarrays• Antibodies• DNA (or RNA) as probes• Specific proteins

Problems:1.proteins must retain their 3-D structure and function2.proteins may interact with multiple targets

ProteomicsProtein microarrays• Antibodies• DNA (or RNA) as probes• Specific proteins

Problems:1.proteins must retain their 3-D structure and function2.proteins may interact with multiple targets3.binding kinetics and affinities differ between proteins, and

are more difficult to standardize than hybridization times

MetabolomicsIdentifying all the metabolites in a given tissue• GC/MS for non-polars• LC/MS for polars

MetabolomicsIdentifying all the metabolites in a given tissue• GC/MS for non-polars• LC/MS for polarsAltered levels of metabolites are often earliest clues to disease

MetabolomicsIdentifying all the metabolites in a given tissue• GC/MS for non-polars• LC/MS for polarsAltered levels of metabolites are often earliest clues to diseaseMost sensitive measures of differences between organisms• Eg parents and hybrids