Date of Award

Fall 2003

Project Type

Dissertation

Program or Major

Plant Biology

Degree Name

Doctor of Philosophy

First Advisor

Estelle M Hrabak

Abstract

In plants, calcium-dependent protein kinases (CDPKs) are the predominant calcium-stimulated kinases and are known to be involved in many cellular processes. CDPK enzymatic activity previously has been detected in many locations in plant cells, including the membrane fraction. However, little is known about the subcellular locations of individual CDPKs or the mechanisms involved in targeting them to those locations. Arabidopsis contains 34 genes that are predicted to encode CDPKs and 28 of the predicted CDPK proteins have potential myristoylation motifs at their amino termini. Myristate is a 14-carbon saturated fatty acid that is attached co-translationally to the amino-terminal glycine of a nascent protein. Myristoylation can facilitate membrane binding and/or protein-protein interactions.

In these studies, Arabidopsis CDPK isoforms AtCPK1, AtCPK2, AtCPK5 and AtCPK6 have been investigated and the subcellular membrane location of each isoform, as well as the role that myristoylation plays in membrane association, have been addressed. An in vitro coupled transcription/translation system was used to demonstrate that AtCPK1, AtCPK2, AtCPK5 and AtCPK6 can be myristoylated and a mutation of the myristoylation site from glycine to alanine (G2A) in AtCPK2 and AtCPK5 prevented myristoylation in vitro. Subcellular localization studies were conducted using both aqueous two-phase partitioning and sucrose density gradient fractionation of plant microsomes. AtCPK1 and AtCPK2 are associated with the endoplasmic reticulum and AtCPK5 and AtCPK6 are associated with the plasma membrane. Disruption of the myristoylation site (G2A mutation) decreased CDPK membrane binding. Using a translational fusion with the beta-glucuronidase (GUS) reporter gene, I have demonstrated that the amino terminal region of AtCPK2 and AtCPK5 contains sufficient information for correct membrane localization.

My results indicate that myristoylation is important in membrane association of CDPKs, that different CDPK isoforms are targeted to different subcellular membrane locations and that the amino terminal region of CDPKs contains specific subcellular targeting information.

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