1. Functional Conservation of Calreticulin in Euglena gracilis Kym Craft Rick Mohanty 05 October 2005

2. What is calreticulin? High capacity, low affinity Ca2+ binding protein High capacity, low affinity Ca2+ binding protein Can hold 20 moles of Ca2+ Can hold 20 moles of Ca2+ Molecular Mass of 50-60 kDa Molecular Mass of 50-60 kDa Localized in ER of eukaryotes (eg. E. Hux) Localized in ER of eukaryotes (eg. E. Hux) Integral to signal transduction pathways involving Ca2+ as second messenger Integral to signal transduction pathways involving Ca2+ as second messenger

3. Why in the ER? Calreticulin is characteristically found in the ER Calreticulin is characteristically found in the ER Possesses short signal peptide Possesses short signal peptide Possesses KDEL retention signal Possesses KDEL retention signal Ideal location for signal transduction pathways Ideal location for signal transduction pathways Converting one signal/stimulus into another Converting one signal/stimulus into another Influences how cell can react and respond to environment. Influences how cell can react and respond to environment.

4. Euglena gracilis Unicellular eukaryotic protist Unicellular eukaryotic protist Branched off relatively early in eukaryotic evolution Branched off relatively early in eukaryotic evolution Plastids have three outer membranes instead of two. Plastids have three outer membranes instead of two. Suggests engulfment of eukaryote, which partially integrated into cell structure. Suggests engulfment of eukaryote, which partially integrated into cell structure.

5. Kinetoplastid Chlorophyte Kinetoplastid is an ancestor of Euglena Kinetoplastid engulfs chlorophyte, and incorporates some of its DNA, also incorporates chloroplast Did calreticulin come from symbiont or host? Was the symbiont a chlorophyte at all? Current Euglena

6. Questions Posed Is the Euglena protein calreticulin? Is the Euglena protein calreticulin? How far back can components of Ca2+ homeostasis be traced within contemporary eukaryotes? How far back can components of Ca2+ homeostasis be traced within contemporary eukaryotes? What is the evolutionary order of appearance of calreticulin? What is the evolutionary order of appearance of calreticulin? If a host eukaryote engulfed a photosynthetic eukaryote, would components of the symbiont be incorporated into the host’s Ca2+ machinery? If a host eukaryote engulfed a photosynthetic eukaryote, would components of the symbiont be incorporated into the host’s Ca2+ machinery?

7. Cell Disruption Euglena cells were grown, then disrupted by: Euglena cells were grown, then disrupted by: French Press: 10,000 psi on target cells French Press: 10,000 psi on target cells Acid-Washed Glass Beads: After cells were initially broken, organelles were kept intact by this method. Acid-Washed Glass Beads: After cells were initially broken, organelles were kept intact by this method.

8. Protein Purification Acidic Ca2+ proteins were isolated by Ammonium Sulphate Precipitation. Acidic Ca2+ proteins were isolated by Ammonium Sulphate Precipitation. Followed by DEAE-Cellulose chromotography Followed by DEAE-Cellulose chromotography Separation technique based on ion exchange Separation technique based on ion exchange

9. Cell Fractionation Organelles separated by Sucrose Density Gradient Centrifugation at 100,000g Organelles separated by Sucrose Density Gradient Centrifugation at 100,000g Organelles sediment in layer that matches their own density. Organelles sediment in layer that matches their own density.

10. Biochemical Techniques - Electrophoresis Two Dimensional Electrophoresis Two Dimensional Electrophoresis Separates by molecular mass and pH (4-6.5) Separates by molecular mass and pH (4-6.5) SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis SDS-PAGE - Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Separates proteins, and can estimate molecular mass Separates proteins, and can estimate molecular mass

11. Biochemical Techniques – Immunoblot Analysis Blots were incubated with: Blots were incubated with: Antibodies against rabbit calreticulin Antibodies against rabbit calreticulin Antibodies against spinach calreticulin Antibodies against spinach calreticulin Cross-Reacted with spinach, no reaction with rabbit Cross-Reacted with spinach, no reaction with rabbit 1.Euglena in spinach antiserum 2.Spinach in spinach antiserum

12. Molecular Techniques Amplification of calreticulin probe: PCR of degenerate nucleotides Amplification of calreticulin probe: PCR of degenerate nucleotides Probe: Detailed piece of DNA, chemically labeled and used to locate sequences. Probe: Detailed piece of DNA, chemically labeled and used to locate sequences. Redundancy in genetic code, multiple codons. Contain different triplets, yet code same amino acid Redundancy in genetic code, multiple codons. Contain different triplets, yet code same amino acid The product was 260bp, and it was amplified again The product was 260bp, and it was amplified again

13. Molecular Techniques This product was purified, then transformed into E. coli yielding the plasmid pPCRcalrH This product was purified, then transformed into E. coli yielding the plasmid pPCRcalrH A specific fragment Cla I-XhoI, was isolated by electrophoresis, then sequenced. A specific fragment Cla I-XhoI, was isolated by electrophoresis, then sequenced. BLAST searches and ClustalW were used to aid in sequence analysis BLAST searches and ClustalW were used to aid in sequence analysis

14. Electrophoresis Analysis Lane 1 shows a 56kDa protein Lane 2 shows a 56kDa protein that can readily bind Ca2+ Lane 3 shows 56kDa protein with acidic character All characterisic of calreticulin

15. Positively Calreticulin All known calreticulins possess sequence DCGGGY All known calreticulins possess sequence DCGGGY Sequence analysis of Euglena cDNA yielded 5 matches for calreticulin Sequence analysis of Euglena cDNA yielded 5 matches for calreticulin Calreticulin is known not to phosphorylate Calreticulin is known not to phosphorylate Euglena did not phosphorylate by protein kinase CK2 Euglena did not phosphorylate by protein kinase CK2

17. Protein Localized in ER Sucrose Density Centrifugation showed results indicative of ER cDNA analysis showed calreticulin signal peptide in N-Terminus Also showed KDEL retention signal in C-terminus Indicative of calreticulin, and localization in the ER

18. Symbiont or Host? Previous studies have shown that Euglena arose through secondary symbiosis. Previous studies have shown that Euglena arose through secondary symbiosis. Euglena showed genes of kinetoplastid and chlorophyte Euglena showed genes of kinetoplastid and chlorophyte Phylogenetic analysis shows Euglena calreticulin branches off from the Leishmania donovani, a kinetoplastoid. Phylogenetic analysis shows Euglena calreticulin branches off from the Leishmania donovani, a kinetoplastoid. The Ca2+ apparatus derived from the host not the symbiont. The Ca2+ apparatus derived from the host not the symbiont. cDNA analysis (KDEL, signal peptide), and phylogenetic analysis suggest that the Ca2+ mechanism was already in place at the time of divergence. cDNA analysis (KDEL, signal peptide), and phylogenetic analysis suggest that the Ca2+ mechanism was already in place at the time of divergence.

19. Calreticulin Phylogenetic Tree