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. 1990 Nov;2(11):1091–1106. doi: 10.1105/tpc.2.11.1091

In Vitro Processing of Aleurain, a Barley Vacuolar Thiol Protease.

BC Holwerda 1, NJ Galvin 1, TJ Baranski 1, JC Rogers 1
PMCID: PMC159957  PMID: 12354950

Abstract

Aleurain, originally described from its cDNA as a thiol protease [Rogers, J.C., Dean, D., and Heck, G.R. (1985). Proc. Natl. Acad. Sci. USA 82, 6512-6516], is characterized here as a glycoprotein that is targeted to a distinct vacuolar compartment in aleurone cells. Monospecific antibodies to a bacterial trpE-aleurain fusion protein were used to show that aleurain is made as a 42-kilodalton (kD) proenzyme (proaleurain) that is proteolytically processed in a post-Golgi compartment in two steps to form a 32-kD protein. The first processing step is the discrete loss of 9 kD from proaleurain to yield a 33-kD intermediate that is further processed by the gradual loss of 1 kD resulting in mature 32-kD aleurain. Using proaleurain secreted from Xenopus oocytes as a substrate, we established an in vitro system using aleurone cell extracts that correctly processes proaleurain to a stable protein that is indistinguishable from native barley aleurain as judged by partial digestion with staphylococcal V8 protease. Proaleurain is not capable of self-cleavage in the absence of aleurone cell extracts and mature aleurain appears not to participate in processing in vitro.

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Selected References

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