The trans-Golgi network (TGN) is an important organelle for protein transport at the post-Golgi network, which functions as a sorting station that directs cargo proteins to a variety of destinations including post-Golgi compartments and the extracellular space. However, the functions and dynamics of the TGN in plant cells have not been well understood yet. To elucidate the dynamics of the plant TGN, we established transgenic plants expressing green fluorescent protein (GFP)-SYP43, the ortholog of Tlg2/syntaxin16, which is localized to the TGN in yeast and mammalian cells, under the control of the native promoter as a TGN marker. Observation by confocal laser scanning microscopy and super-resolution confocal live imaging microscopy revealed two types of TGN in Arabidopsis root: the GA-TGNs (Golgi-associated TGNs), located on the trans-side of the Golgi apparatus, and the GI-TGNs (Golgi-released independent TGNs), located away from the Golgi apparatus and behaving independently. The GI-TGNs is derived from a population of GA-TGNs by segregation, although the core of the GA-TGN remains even after the generation of the GI-TGN. We further found that the abundance of the GI-TGNs differs between observed tissues. Our results indicate that the dynamic features of the TGN in plant cells differ from those of animal and yeast cells.

Significance The Golgi apparatus functions as the central station of membrane traffic in cells. A series of Rab GTPases, which control various steps in membrane traffic, act consecutively during the course of Golgi maturation. Here, we report that Ypt6, a Rab6 homologue in yeast, resides temporarily at the Golgi and dissociates into the cytosol upon arrival of Ypt32, another Rab GTPase functioning in the late Golgi. We have found that Gyp6, a putative GTPase-activating protein for Ypt6, specifically interacts with Ypt32 as an effector. Taken together with the previously proposed Rab cascade within the Golgi, we propose that multiple Rab cascades interact at the intersection of secretory and endosomal pathways and play significant roles in traffic within and around the Golgi apparatus.

To describe trafficking of secretory cargo within the Golgi apparatus, the cisternal maturation model predicts that Golgi cisternae change their properties from cis to trans while cargo remains in the cisternae. Cisternal change has been demonstrated in living yeast Saccharomyces cerevisiae; however, the behavior of cargo has yet to be examined directly. In this study, we conducted simultaneous three-color and four-dimensional visualization of secretory transmembrane cargo together with early and late Golgi resident proteins. We show that cargo stays in a Golgi cisterna during maturation from cis-Golgi to trans-Golgi and further to the trans-Golgi network (TGN), which involves dynamic mixing and segregation of two zones of the earlier and later Golgi resident proteins. The location of cargo changes from the early to the late zone within the cisterna during the progression of maturation. In addition, cargo shows an interesting behavior during the maturation to the TGN. After most cargo has reached the TGN zone, a small amount of cargo frequently reappears in the earlier zone.

Formation of ascospores in the yeast Saccharomyces cerevisiae is driven by an unusual cell division in which daughter nuclei are encapsulated within de novo-formed plasma membranes, termed prospore membranes. Generation of viable spores requires that cytoplasmic organelles also be captured along with nuclei. In mitotic cells segregation of mitochondria into the bud requires a polarized actin cytoskeleton. In contrast, genes involved in actin-mediated transport are not essential for sporulation. Instead, efficient segregation of mitochondria into spores requires Ady3p, a component of a protein coat found at the leading edge of the prospore membrane. Other organelles whose mitotic segregation is promoted by actin, such as the vacuole and the cortical endoplasmic reticulum, are not actively segregated during sporulation but are regenerated within spores. These results reveal that organellar segregation into spores is achieved by mechanisms distinct from those in mitotic cells.