Introduction: stepping into the science

What kind of research is being done on the project?

What is an Arabidopsis plant?

How does the ABE workshop fit in?

What kind of results might you expect?

• What the heck does that mean?

National Sciences Foundation (NSF) funds the ABE workshop through a research project entitled:

“Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and

Redox Regulatory Networks”

What kind of research is being done on the project?

“Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and

Redox Regulatory Networks”

Functional Genomics:

• New field of biological science

• Genetics-based

• Genome: all of the genes encoded in DNA in a living organism.

• Function: Conduct research to figure out what the genes are doing.

• What proteins do they encode and what jobs in the cell are they responsible for?

What jobs do the proteins do in a cell?

• 1. Structure: hold things up

• 2. Enzymes: activity make and burn energy.

Stimulate growth and biomass production.

• 1000’s different enzymes -> unique activities

• Figure out their activities.

ENZA -----------> B

• Where the enzyme is located in the cell?

• Do they need other protein partners to do their job?

“Functional Genomics of Protein Disulfide Isomerase Gene Family: Unraveling Protein Folding and

Redox Regulatory Networks”

ENZYME: Protein Disulfide Isomerase

• Protein = chain of amino acids. Results from de-coding the mRNA sequence transcribed from DNA.

• Disulfide: “Two sulfurs”

The amino acid containing sulfur is cysteine

Protein with 2 cysteines

|SH

|SH

ENZYME: Protein Disulfide Isomerase

• Isomer: Different substances that have the same components.

= Different molecules with same chemical formula • Alter chemical bonding --> different “shapes” --> activities and functions.

•Isomerase: an enzyme that can make different molecular shapes out of the same substance.

|SH

|SH

Protein Disulfide Isomerase (PDI) can make different protein shapes based on altered

sulfur bonding

-S-S-

An Isomer with new activity !

Proteins do not do their job unless they are folded correctly

• So, PDIs fold other proteins correctly in cells.

• A major responsibility for keeping cells normal, development, metabolism and growth.

Protein Disulfide Isomerase (PDI) Gene Family

• Study all the PDIs in the genome of a small plant.

•All the PDIs in the same related family. • but they go off and have different jobs at various locations in the cell.

PDI Protein folding- oxidoreductase

PDI

= cys

Inactive state Active state

• Oxidation of 2 cysteine sulfhydryls --> disulfide bridge– Remove 2 electrons and 2 H+

• Reduction --> breaks bridge --> 2 sulfhydryls– Add 2 electrons and 2 H+

All proteins have to fold to proper states

Chemical Mechanism

• But what about plants????

In Yeast and humans - PDIs located in the endoplasmic reticulum (ER)

Arabidopsis thaliana Plants

• First plant to have its genome completely sequenced– Smallest Plant Genome known

-> 26,000 genes but makes a plant!!

– 40 days from seed to seed.

– Easily genetically engineered.

– Easy to knock out genes to see what they do.

Arabidopsis

There are 11 different PDI’s in Arabidopsis plantsPrimary Structure of the PDI family in Arabidopsis

KEY

thioredoxin domain =PWCghC (dithiol)

thioredoxin domain =PWCARS (monothiol)

KDEL -endoplasmic reticulum retention motif

pdi1

pdi2

pdi3

pdi4

pdi5

pdi6

pdi7

pdi8

pdi9

pdi10

pdi11

KDEL

KDEL

KDEL

KDEL

KDEL

KDEL

KDEL

Endo. Retic. signal peptide

chloroplasttransit peptide

mitochondrialtransit peptide

= 25 amino acids

Research goals of workshop

Learn some recombinant DNA methods

Mapping genes that have been tagged by a T-DNA

Microscopy to locate where PDIs are using PDI-seeking antibodies.

What kind of results might you expect?

Ooops, Not this….

Any kind of result is a success Learn by doing !!!

Have fun while you learn !

Nothing has to work perfectly to be a valuable learning experience.

M 1-a 1-b 3-Ⅰ 3-Ⅱ 3-Ⅲ 4-Ⅰ 4-Ⅱ 4-Ⅲ W.T

T-DNA Mapping

PDI antisera binds to chloroplasts of Arabidopsis

Localization to chloroplasts and cell wall

Localization to chloroplasts: Red chlorophyll fluorescence

Alignment of Arabidopsis PDI2 with Chlamydomonas PDIP D I 2 1 - - - - - - - - - - - - - - M Y K S P L T L L T L L T I C F G F F D L S S A L Y G S S S P - - - - V V Q L T A S N F K SP D I - C h l y 1 M N R W N L L A L T L G L L L V A A P F T K H Q F A H A S D E Y E D D E E D D A P A A P K D D D V D V T V V T V K N W Dc o n s e n s u s 1 m n r w n l l a l t l g l l m P T f D d d d v V l

P D I 2 4 3 K V L N S N G V V L V E F F A P W C G H C K A L T P T W E K V A N I L K G V A T - - - V A A I D A D A H Q S A A Q D Y GP D I - C h l y 6 1 E T V K K S K F A L V E F Y A P W C G H C K T L K P E Y A K A A T A L K A A A P D A L I A K V D A T Q E E S L A Q K F Gc o n s e n s u s 6 1 l L V E F f A P W C G H C K L P w K A L K g A d a l v A i D A S A Q y G

P D I 2 1 0 0 I K G F P T I K V F V P G K A P I D Y Q G A R D A K S I A N F A Y K Q I K G - - - - - - - - - - - - - - - - - - - - - -P D I - C h l y 1 2 1 V Q G Y P T L K W F V D G E L A S D Y N G P R D A D G I V G W V K K K T G P P A V T V E D A D K L K S L E A D A E V V Vc o n s e n s u s 1 2 1 i G f P T i K F V G D Y q G R D A I f K p a v t v e d a d k l k s l e a d a e v v v

P D I 2 1 3 8 - L L S D R L E G K S K P T G G G S K E K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -P D I - C h l y 1 8 1 V G Y F K A L E G E I Y D T F K S Y A A K T E D V V F V Q T T S A D V A K A A G L D A V D T V S V V K N F A G E D R A Tc o n s e n s u s 1 8 1 v L E G T K t e d v v f v q t t s a d v a k a a g l d a v d t v s v v k n f a g e d r a t

P D I 2 1 5 8 - - - - - - - - - - - - - - - - - K S E P S A S V E L N A S N F D D L V I E - S N E L W I V E F F A P W C G H C K K L AP D I - C h l y 2 4 1 A V L A T D I D T D S L T A F V K S E K M P P T I E F N Q K N S D K I F N S G I N K Q L I L W T T A D D L K A D A E I Mc o n s e n s u s 2 4 1 a v l a t d i d t d s l t a f v k s v E N N D l g N I v A l

P D I 2 2 0 0 P E W K R A A K N L Q G K V K L G H V N - - C D V E Q S I M S R F K V Q G - - F P T I L V F G P D K S S P Y P Y E G A RP D I - C h l y 3 0 1 T V F R E A S K K F K G Q L V F V T V N N E G D G A D P V T N F F G L K G A T S P V L L G F F M E K N K K F R M E G E Fc o n s e n s u s 3 0 1 w k A K G v V N n e D i F v G a t P i L F d K y E G

P D I 2 2 5 6 S A S A I E S F A S E L V E S S A G P V E V T E L T G P D V M E K K C G S A A I C F I S - - F L P D I L D S K A E G R NP D I - C h l y 3 6 1 T A D N V A K F A E S V V D G T A Q A V L K S E A I P E D P Y E D G V Y K I V G K T V E S V V L D E T K D V L L E V Y Ac o n s e n s u s 3 6 1 s A i F A l V e s A V t E D E i s v L d D E

P D I 2 3 1 4 K Y L E M L L S V A E K F K K Q P Y S F M W V A A V T Q M D L E K R V N V G G Y G Y P A M V A M N V K K G V Y A P L K SP D I - C h l y 4 2 1 P W C G H C K K L E P I Y K K L A K R F K K V D S V I I A K M D G T E N E H - - - - P E I E V K G F P T I L F Y P A G Sc o n s e n s u s 4 2 1 y v f K K F V V l e N g y g y P m v y P S

P D I 2 3 7 4 A F E L Q H L L E F V K D A G T G G K G N V P M N G T P E I V K T K E W D G K D G E L I E E D E F S L D E L M G G D D AP D I - C h l y 4 7 7 D R T P I V F E G G D R S L K S L T K F I K T N A K I P Y E L P K K G S D G D E G T S D D K D - - - - - - - K P - - - -c o n s e n s u s 4 8 1 k t K P v K D G d G e D e f s l d e l g d d a

P D I 2 4 3 4 V G S K D E LP D I - C h l y 5 2 6 A S D K D E Lc o n s e n s u s 5 4 1 K D E L

M N R W N L L A L T L G L L D D D V

D A L

P A V T V E D A D K L K S L E A D A E V V V

V T E D V V F V Q T T S A D V A K A A G L D A V D T V S V V K N F A G E D R A T

A V L A T D I D T D S L T A F V K G

G G

A TN E

S V

G Y G Y

E F S L D E L G D D A

K S EK S E

PDI2 is processed by ER microsomes indicating it has a N-terminal signal peptide destined for ER or secrettion

0 20 60 90 min

Transmission electron microscopy: Immunolocalization

TEM: Immunolocalization to periphery of starch in chloroplasts

TEM: Asymmetry starch biogenesis