4 Primer on Protein Targeting

8/2/2019 4 Primer on Protein Targeting

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Protein Targeting in Plant

Biofactories

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This primer is based on a talk by Dr. Jaimie Schnell and ismeant to be a reminder of what you took in previous years.Developed for plants the ideas apply to all eucaryotes.


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Plant Biofactories

Cheaper than mammalian, insect, or

prokaryotic systems

Agricultural infrastructure already in place

Easy to scale-up

No human pathogens, endotoxins in plants

Correct folding, modifications

High volume Direct use

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High level expression is essential

for economic recombinant proteinproduction

Offset costs of production, purification

Compete with other sources

Ensure efficacy

Streatfield 2007

To dateFew small-scale products have reached market

Not produced economically on large-scale

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Improving Plant Biofactories

Tissue-specific expression

oil bodies, seeds, tubers

Plastid transformation

Molecular approaches

Protein targeting

Higher yield

Long-term stability Reduced toxicity

Correct processing

Streatfield 2007

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Protein Targeting in Plants

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How do plants sort proteins?

Targeting information is contained in

peptide signaling sequences

Signals are recognized by receptors that

direct them to the target organelle

Translocation via:

Transmembrane channels

Vesicles

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The Secretory System(cartoon version)

Endoplasmic

Reticulum

Golgi network

Vacuole

PM

Vesicle

PlasmaMembrane

Prevacuolar

Compartment

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Protein Targeting: examples & mechanisms:

Via cloning we create fusion proteins that direct yourprotein to the desired location.

The signal peptides come from previously identified

genes.

The primary locations for targeting are members of theendomembrane system (ER, Golgi, Vacuole) and outside

of the cell (in plants the apoplast).

Experimentally how do we do this??

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Protein Targeting: targeted proteins with GFP

GFP gene

Endoplasmic ReticulumGFP gene

GFP gene

=ER signal =ER retention signal

Predicted Location?

Could you do this on a

genomics scales?

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For example the endoplasmic reticulum

Endoplasmic Reticulum

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ApoplastApoplast

For example outside of the cell (apoplast)

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Arabidopsis protoplast

Red = chloroplasts;

Green = mitochondria

Mitochondrian Chloroplast

For example organelles

Arabidopsis leafvacuole

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http://www.google.ca/imgres?imgurl=http://www.lifesci.sussex.ac.uk/home/Julian_Thorpe/vacuole.jpg&imgrefurl=http://www.lifesci.sussex.ac.uk/home/Julian_Thorpe/vacuole.htm&usg=__9aFEseelZxHUXP8SIZ5j4fwgHEI=&h=334&w=400&sz=87&hl=en&start=3&zoom=1&tbnid=0OExgHapiGIeqM:&tbnh=104&tbnw=124&prev=/images%3Fq%3Dvacuole%2BEM%26hl%3Den%26gbv%3D2%26tbs%3Disch:1&itbs=1


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What are the locations we are going to describe?-nucleus

Nucleus

BIO1140 Unit 3 Protein targeting


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The Secretory System

ApoplastDefault pathway

Endoplasmic reticulumConserved C-terminal H/KDEL sequence

Retention/recovery signal

ER TS

-HDELER TS

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The endoplasmic reticulum target signal

N-terminal

Conserved structure

Directs co-translational import into the

endoplasmic reticulum

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Vacuole

Sequence Specific Vacuolar Sorting

Determinant

Typically N-terminal

Conserved NPIR consensus sequence

C-Terminal Vacuolar Sorting DeterminantNo conserved sequence

Hydrophobicity important

ctVSDER TS

ssVSDER TS

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The Chloroplast

N-terminal

transit signal

Imported into

stroma post-translationally

May have

additional

signals for

further sorting

Jarvis and Robinson 200417


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The Apoplast

Benefits:Higher yields

Post-translationallymodified

Example:Accumulation of phytase in tobacco leaves

(Verwoerd et al. 1995)Reached levels of 14.4% phytase per mg of total

soluble protein

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The Endoplasmic Reticulum

Benefits:Higher yields

Example:Arabidopsis plants expressing syntheticanalogue of spider dragline silk protein (Yanget al. 2005)

As high as 18% total soluble protein in seeds whentargeted to the ER

Highest yield when targeted to the cytoplasmwas 1.4%

C 1 2

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The Chloroplast

Benefits:Higher yield

Isolation of potentially toxiccompounds

Example:Expression of cholesterol oxidase in tobacco

(Corbin et al. 2001)Chloroplast targeted plants were indistinguishable

from wild-type

Non-targeted plants exhibited several phenotypicabnormalities

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Relationship of the section Protein Targeting tothe NSERC projects

Can lead to higher accumulation of your protein

Can protect the host from the effect of the protein

Modifications can be human-like

(but this could be a problem!)

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4 Primer on Protein Targeting