Myristoylation And Plasma Membrane Binding Regulate Tobacco NtRab5b Transcription

Tobacco (Nicotiana tobacum) NtRab5b, a plant-unique small GTP-binding protein, is localized to the endocytosis pathway and involved in the response to salt stress in seedlings.This study shows that the N-terminal myristoylation signal mediated NtRab5b association with cellular membranes. The negative mutant (G2A) was mis-targeted to cytosol and nuclei and resulted in a significant up-regulation of NtRab5b(G2A) mRNA . The differential expression of NtRab5b and NtRab5b(G2A) exists both in the CaMV35S and JcUEP promoters transgenic BY-2 cells, besides fusing with GFP have not changed this phenomenon. These results suggest that the transcriptional up-regulation of NtRab5b(G2A) in transgenic cells is due to the mutation of the N-terminal myristoylation site Gly2.Next we find that both the N-terminal palmitoylation site and the GTPase characteristic were also critical to the transcription of NtRab5b in transgenic cells. NtRab5b(C3S)-GFP which has Gly2 where N-myristoylation occurs but cannot be a substrate for palmitoylation, was localized mainly on the ER and its transcription is up-regulated contrast to NtRab5b-GFP, but mutation of the Gly at position 2 cannot increase the transcription of myristoylation and palmitoylation negative mutant NtRab5b(G2A/C3S)-GFP. GTPase negative mutant NtRab5b(Q92L)-GFP which is expected to be stabilized GTP-bound state, was observed on vacuole membrane and small dots but without on plasma membrane. Transcriptional expression analysis shows that NtRab5b(Q92L) and NtRab5b(Q92L)-GFP were up-regulated contrast to NtRab5b and NtRab5b-GFP respectively, but mutation of the Gly at position 2 has not affected their transcription. Thus, the absence affinity with plasma membrane is responsible for the transcriptional up-regulation of domain-negative mutant gene NtRab5b (G2A, C3S or Q92L).Then we have research the regulatory effect of N-termini sequences in the NtRab5b transcription. The results suggest that N-terminal 40 amino acids tagged by GFP (MyrN40-GFP) resides mainly on the plasma and the mutation of Gly2 (MyrN40(G2A)-GFP) have increased noticeably its mRNA expression, but when deleting N-terminal sequences to 20-, 11-, 6- amino acids, mutation of the Gly at position 2 cannot increase their transcriptions. Therefore, the 20~40 amino acid sequence is critical to the regulatory effect of myristoylation in the NtRab5b transcription.Finally, combined the GFP subcellular localization and expression in the 35S:GFP, we concluded that NtRab5b-GFP and MyrN40-GFP likely possess the ability of feedback inhibition that the myristoylation protein can inhibit its self-gene expression by the association with plasma membrane mediated by the myristoylation on the Gly2.