Visualizing Reversible Cisternal Stacking in Budding Yeast Pichia pastoris.

Cisternal stacking is reversible, initiated at the "cis" side of the Golgi, and gets undone at the "trans" side in a continuous cycle in tune with the cisternal maturation. TGN peeling is a hallmark of such reversible cisternal stacking, but its visualization is challenging. In wild-type cells, TGN peeling of Golgi stack happens at a lower frequency, but the event itself occurs very rapidly, making it difficult to detect by microscopy. However, we have documented that TGN peeling becomes frequent in mutants of factors that play a role in reversible cisternal stacking, such as the GRIP domain Golgin PpImh1, Arl3, or Arl1 GTPase. In the present context, we describe the quantitative live microscopic methodology to visualize the TGN peeling effect in Pichia pastoris.

The golgin PpImh1 mediates reversible cisternal stacking in the Golgi of the budding yeast Pichia pastoris.

The adhesive force for cisternal stacking of Golgi needs to be reversible - to be initiated and undone in a continuous cycle to keep up with the cisternal maturation. Microscopic evidence in support of such a reversible nature of stacking, in the form of 'TGN peeling,' has been reported in various species, suggesting a potential evolutionarily conserved mechanism. However, knowledge of such mechanism has remained sketchy. Here, we have explored this issue in the budding yeast Pichia pastoris which harbors stacked Golgi. We observed that deletion of GRIP domain golgin P. pastoris (Pp)IMH1 increases the peeling of late cisterna, causing unstacking of the Golgi stack. Our results suggest that the PpImh1 dimer mediates reversible stacking through a continuous association-dissociation cycle of its GRIP domain to the middle and late Golgi cisterna under the GTP hydrolysis-based regulation of Arl3-Arl1 GTPase cascade switch. The reversible cisternal stacking function of PpImh1 is independent of its vesicle-capturing function. Since GRIP domain proteins are conserved in plants, animals and fungi, it is plausible that this reversible mechanism of Golgi stacking is evolutionarily conserved.This article has an associated First Person interview with the first author of the paper.