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

Abstract

The adhesive force for cisternal stacking of Golgi needs to be reversibleto 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.

ER arrival sites associate with ER exit sites to create bidirectional transport portals

“…This fragment was digested with EcoRI and SphI and subcloned into pIB1 ( Sears et al, 1998 ) cut with the same enzymes. BamHI and NotI sites were introduced by site-directed mutagenesis immediately after the start codon, and a cassette encoding msGFP was excised with BamHI and NotI and inserted ( Jain et al, 2019 ). This plasmid was linearized with StuI and integrated at the HIS4 locus.…”

“…A pUC19-ARG4 derivative containing a 3′ portion of the P. pastoris SEC7 coding sequence plus a downstream region ( Bevis et al, 2002 ) was modified by inserting a 6xDsRed.M1 cassette in place of the stop codon ( Losev et al, 2006 ). This plasmid was linearized with XmnI and integrated at the SEC7 locus (( Jain et al, 2018 ); Jain et al, 2019 ).…”

Knockout of RSN1, TVP18 or CSC1‐2 causes perturbation of Golgi cisternae in Pichia pastoris

“…To observe the structural changes in the Golgi it was necessary to generate a visualization method that could indicate whether the Golgi remained stacked or was disrupted. Previous research has shown that it is possible to use two different fluorescent proteins fused to early and late Golgi markers 16,20,22,23 where co‐localization indicates a stacked Golgi. In order to generate such a system, we selected Vrg4p (PAS_chr3_0916), used by Connerly et al, 20 Losev et al, 23 and Jain et al 22 as the early Golgi marker, and fused this to a superfolder GFP (sfGFP).…”

“…Previous research has shown that it is possible to use two different fluorescent proteins fused to early and late Golgi markers 16,20,22,23 where co‐localization indicates a stacked Golgi. In order to generate such a system, we selected Vrg4p (PAS_chr3_0916), used by Connerly et al, 20 Losev et al, 23 and Jain et al 22 as the early Golgi marker, and fused this to a superfolder GFP (sfGFP). Although previous research had used Sec7p (PAS_chr1‐4_0667) as the late Golgi marker, 24 we were interested in observing the impact of this gene on the structural organization of the Golgi.…”

“…Connerly et al 20 previously reported a SEC16 mutant that resulted in temperature‐induced disruption of the Golgi structure when grown at 36.5°C. Additionally, Jain et al 21 identified a GRIP domain Golgin Pplmh1 and later established that a knockout of this gene results in reversible unstacking of the Golgi 22 . As neither of these mutants resulted in permanent unstacking under physiological conditions, we undertook a broader investigation in P. pastoris to identify other factors involved in the formation of Golgi stacks.…”