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Vol. 15, Issue 2, 481-496, February 2004

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The EF-Hand Ca²⁺-binding Protein p22 Plays a Role in Microtubule and Endoplasmic Reticulum Organization and Dynamics with Distinct Ca²⁺-binding Requirements

Albany Medical Center; Center for Cardiovascular Sciences, Albany, New York 12208

Submitted July 17, 2003; Revised October 8, 2003; Accepted November 7, 2003
Monitoring Editor: Reid Gilmore

We have reported that p22, an N-myristoylated EF-hand Ca²⁺-binding protein, associates with microtubules and plays a role in membrane trafficking. Here, we show that p22 also associates with membranes of the early secretory pathway membranes, in particular endoplasmic reticulum (ER). On binding of Ca²⁺, p22's ability to associate with membranes increases in an N-myristoylation-dependent manner, which is suggestive of a nonclassical Ca²⁺-myristoyl switch mechanism. To address the intracellular functions of p22, a digitonin-based "bulk microinjection" assay was developed to load cells with anti-p22, wild-type, or mutant p22 proteins. Antibodies against a p22 peptide induce microtubule depolymerization and ER fragmentation; this antibody-mediated effect is overcome by preincubation with the respective p22 peptide. In contrast, N-myristoylated p22 induces the formation of microtubule bundles, the accumulation of ER structures along the bundles as well as an increase in ER network formation. An N-myristoylated Ca²⁺-binding p22 mutant, which is unable to undergo Ca²⁺-mediated conformational changes, induces microtubule bundling and accumulation of ER structures along the bundles but does not increase ER network formation. Together, these data strongly suggest that p22 modulates the organization and dynamics of microtubule cytoskeleton in a Ca²⁺-independent manner and affects ER network assembly in a Ca²⁺-dependent manner.

Abbreviations used: ER, endoplasmic reticulum; myr-p22, N-myristoylated bacterially expressed p22; myr-p22-E134A, bacterially expressed N-myristoylated EF-3 Ca²⁺-binding mutant; p22-rec, non-myristoylated bacterially expressed p22; APpep2 antibodies, affinity-purified antibodies against an unique p22 peptide, pep2; APp22 antibodies, affinity-purified antibodies against full-length p22.

^* Corresponding author. E-mail address: barrosm{at}mail.amc.edu.

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