Isolation of a tobacco cDNA encoding Sar1 GTPase and analysis of its dominant mutations in vesicular traffic using a yeast complementation system.

The cDNA clone of NtSAR1, a gene encoding the small GTPase Sar1p which is essential for vesicle formation from the endoplasmic reticulum (ER) membrane in yeast, has been isolated from Nicotiana tabacum BY-2 cells. NtSAR1 as well as AtSAR1 cDNA isolated from Arabidopsis thaliana [d'Enfert et al. (1992) EMBO J. 11: 4205] could complement the lethality of the disruption of SAR1 in yeast cells in a temperature-sensitive fashion. They also suppressed yeast sec12 and sec16 temperature-sensitive mutations as yeast SAR1 does. Using this complementation system, we analyzed the phenotypes of several mutations in plant SAR1 cDNAs in yeast cells. The expression of NtSAR1 H74L and AtSAR1 N129I showed dominant negative effect in growth over the wild-type SAR1, which was accompanied by the arrest of ER-to-Golgi transport. Such dominant mutations will be useful to analyze the role of membrane trafficking in plant cells, if their expression can be regulated conditionally.