A Missense Mutation in the <i>NSF</i> Gene Causes Abnormal Golgi Morphology in <i>Arabidopsis thaliana</i>

Abstract

The Golgi apparatus is a key station of glycosylation and membrane traffic. It consists of stacked cisternae in most eukaryotes. However, the mechanisms how the Golgi stacks are formed and maintained are still obscure. The model plant Arabidopsis thaliana provides a nice system to observe Golgi structures by light microscopy, because the Golgi in A. thaliana is in the form of mini-stacks that are distributed throughout the cytoplasm. To obtain a clue to understand the molecular basis of Golgi morphology, we took a forward-genetic approach to isolate A. thaliana mutants that show abnormal structures of the Golgi under a confocal microscope. In the present report, we describe characterization of one of such mutants, named #46-3. The #46-3 mutant showed pleiotropic Golgi phenotypes. The Golgi size was in majority smaller than the wild type, but varied from very small ones, sometimes without clear association of cis and trans cisternae, to abnormally large ones under a confocal microscope.At the ultrastructual level by electron microscopy, queer-shaped large Golgi stacks were occasionally observed. By positional mapping, genome sequencing, and complementation and allelism tests, we linked the mutant phenotype to the missense mutation D374N in the NSF gene, encoding the N-ethylmaleimide-sensitive factor (NSF), a key component of membrane fusion. This residue is near the ATP-binding site of NSF, which is very well conserved in eukaryotes, suggesting that the biochemical function of NSF is important for maintaining the normal morphology of the Golgi.3

AtNSF regulates leaf serration by modulating intracellular trafficking of PIN1 in Arabidopsis thaliana

“…Wild‐type cells contained the typical six stacked cisternae, but the Golgi shape was malformed and smaller in the atnsf‐1 mutant (Figure S6). This is consistent with the previous report (Tanabashi et al, 2018), indicating that AtNSF indeed plays a role in the structure of the Golgi apparatus, and is involved in vesicle trafficking from the Golgi to other endosomes and/or the PM.…”

“…Consistent with the functions of these NSF orthologs, AtNSF also plays an essential role in membrane trafficking. The AtNSF protein is localized to the Golgi apparatus, endosome, and cell plate, and its mutation caused pleiotropic Golgi defects (Figures 4B, C, S6; Tanabashi et al, 2018), suggesting it is critically involved in vesicle trafficking between endomembranes. Defects in Golgi morphology in atnsf‐1 were also revealed by TEM images (Figure S6).…”

“…A defective Golgi phenotype was previously observed in an AtNSF mutant (Tanabashi et al, 2018). To determine the Golgi structure in atnsf-1, we observed the cellular ultrastructure by transmission electron microscopy (TEM).…”

A minimal self-organisation model of the Golgi apparatus

“…The predicted role of the budding and fusion rates is in agreement with the phenotype observed upon deletion of Arf1 (a protein involved in vesicle budding), which decreases the number of compartments and increases their size, particularly that of trans -compartments which seem to aggregate into one major cisterna ( Bhave et al, 2014 ). A comparable phenotype has been observed upon mutation of NSF (a fusion protein), which produces extremely large, but transient, trans -compartments ( Tanabashi et al, 2018 ), thereby increasing the stochasticity of the system. This is consistent with an increase of the fusion rate according to our model (see Appendix 5 for a quantification of the fluctuations in our model).…”

A Minimal Self-organisation model of the Golgi Apparatus

“…The predicted role of the budding and fusion rates is in agreement with the phenotype observed upon deletion of Arf1 (a protein involved in vesicle budding), which decreases the number of compartments and increases their size, particularly that of trans -compartments which seem to aggregate into one major cisterna ( Bhave et al, 2014 ). A comparable phenotype has been observed upon mutation of NSF (a fusion protein), which produces extremely large, but transient, trans -compartments ( Tanabashi et al, 2018 ), thereby increasing the stochasticity of the system. This is consistent with an increase of the fusion rate according to our model (see App.5, p.34 for a quantification of the fluctuations in our model).…”