In vivo modifications of the maize mitochondrial small heat stress protein, HSP22.
A maize (Zea mays L.) small heat shock protein (HSP), HSP22, was previously shown to accumulate to high levels in mitochondria during heat stress. Here we have purified native HSP22 and resolved the protein into three peaks using reverse phase high performance liquid chromatography. Mass spectrometry (MS) of the first two peaks revealed the presence of two HSP22 forms in each peak which differed in mass by 80 daltons (Da), indicative of a monophosphorylation. Phosphorylation of HSP22 by [gamma-(32)P]ATP was also observed in mitochondria labeled in vitro, but not when purified native HSP22 was similarly used, demonstrating that HSP22 does not autophosphorylate, implicating a kinase involvement in vivo. Collisionally induced dissociation tandem MS (CID MS/MS) identified Ser(59) as the phosphorylated residue. We have also observed forms of HSP22 that result from alternative intron splicing. The two HSP22 proteins in the first peak were approximately 57 Da larger than the two HSP22 proteins in the second peak. MS analysis revealed that the +57-Da forms have an additional Gly residue directly N-terminal of the expected Asp(84), which had been converted to an Asn residue. These results are the first demonstrations of phosphorylation and alternative intron splicing of a plant small HSP.
Lund
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School of Biological Sciences and the Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588-0666, USA.
Rhoads
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DM
Lund
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AL
Cerny
R L
RL
Elthon
T E
TE
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D001483:Base Sequence; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D018076:DNA, Complementary / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D007438:Introns; D013058:Mass Spectrometry; D008928:Mitochondria / Q000737:chemistry*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry*
D018076:DNA, Complementary; C096801:HSP22 protein, plant; D006360:Heat-Shock Proteins; D001224:Aspartic Acid; D012694:Serine; D001216:Asparagine