Different sensitivity to wortmannin of two vacuolar sorting signals indicates the presence of distinct sorting machineries in tobacco cells

Ken Matsuoka, Diane C. Bassham, Natasha V. Raikhel, Kenzo Nakamura

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210 Citations (Scopus)

Abstract

Vacuolar matrix proteins in plant cells are sorted from the secretory pathway to the vacuoles at the Golgi apparatus. Previously, we reported that the NH2-terminal propeptide (NTPP) of the sporamin precursor and the COOH- terminal propeptide (CTPP) of the barley lectin precursor contain information for vacuolar sorting. To analyze whether these propeptides are interchangeable, we expressed constructs consisting of wild-type or mutated NTPP with the mature part of barley lectin and sporamin with CTPP and mutated NTPP in tobacco BY-2 cells. The vacuolar localization of these constructs indicated that the signals were interchangeable. We next analyzed the effect of wortmannin, a specific inhibitor of mammalian phosphatidylinositol (PI) 3- kinase on vacuolar delivery by NTPP and CTPP in tobacco cells. Pulse-chase analysis indicated that 33 μM wortmannin caused almost complete inhibition of CTPP-mediated transport to the vacuoles, while NTPP-mediated transport displayed almost no sensitivity to wortmannin at this concentration. This indicates that there are at least two different mechanisms for vacuolar sorting in tobacco cells, and the CTPP-mediated pathway is sensitive to wortmannin. We compared the dose dependencies of wortmannin on the inhibition of CTPP-mediated vacuolar delivery of proteins and on the inhibition of the synthesis of phospholipids in tobacco cells. Wortmannin inhibited PI 3- and PI 4-kinase activities and phospholipid synthesis. Missorting caused by wortmannin displays a dose dependency that is similar to the dose dependency for the inhibition of synthesis of PI 4-phosphate and major phospholipids. This is different, however, than the inhibition of synthesis of PI 3- phosphate. Thus, the synthesis of phospholipids could be involved in CTPP- mediated vacuolar transport.

Original languageEnglish
Pages (from-to)1307-1318
Number of pages12
JournalJournal of Cell Biology
Volume130
Issue number6
DOIs
Publication statusPublished - Sep 1995

All Science Journal Classification (ASJC) codes

  • Cell Biology

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