N-Linked Glycosylation Modulates Golgi-Independent Vacuolar Sorting Mediated by the Plant Specific Insert

Abstract

In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlike its native glycosylated form, is able to bypass ER-to-Golgi blockage and accumulate in the vacuole. This is also true when the PSI domain is analyzed in the context of the full-length cardosin. Regardless of opening exciting research gaps, the results obtained so far need a more comprehensive study of the mechanisms behind this unconventional direct sorting to the vacuole.

Living on the edge: the role of Atgolgin‐84A at the plant ER–Golgi interface

“…Trafficking to the vacuole can be either through a Golgi‐dependent route or a Golgi‐independent route directly from the ER in precursor accumulating vesicles and fuse directly to the protein storage vacuole (Hara‐Nishimura et al ., 1998; Vitale & Raikhel, 1999; Chrispeels & Herman, 2000). The Golgi‐independent route has been described for the plant‐specific insert in tobacco cells when the Golgi‐mediated route is impaired by the expression of dominant‐negative mutant of GTPases, for example, the Sar1‐GTP locked version (Pereira et al ., 2013; Vieira et al ., 2019). The pre‐ cis‐ Golgi also contains proteins with specific roles in protein sorting (Ito et al ., 2018), suggesting that there might be a recognition of cargo before the entry in the cis ‐Golgi body.…”

“…The pre‐ cis‐ Golgi also contains proteins with specific roles in protein sorting (Ito et al ., 2018), suggesting that there might be a recognition of cargo before the entry in the cis ‐Golgi body. This could explain the Golgi bypass reported for some vacuole‐targeted proteins (Pereira et al ., 2013; Vieira et al ., 2019).…”

Abiotic Stress Triggers the Expression of Genes Involved in Protein Storage Vacuole and Exocyst-Mediated Routes

“…Transport and compartmentalisation events of a large amount of material without the involvement of the classic vesicle traffic have escaped full characterisation. One example is the recently characterised vacuolar route mediated by the PSI (Plant Specific Insert), which follows an alternative pathway independent of the Golgi [ 15 ]. This process is generically described as unconventional trafficking, and several examples have been reported without a deep mechanism investigation [ 16 , 17 ].…”

Abiotic Stress Upregulates the Expression of Genes Involved in PSV and Autophagy Routes

“…Transport and compartmentalization events of large amount of material out of the classic vesicle traffic route have escaped full characterization. One example is the recently characterized vacuolar route mediated by the PSI (Plant Specific Insert), that was shown to follow an alternative pathway, independent of the Golgi [12]. The PSI is a unique domain, with approximately 100 amino acids, present in the primary structure of some plant aspartic proteinases (APs) [13,14], which plays a role in germination, senescence, organism defense and protein turnover [15] This sequence is responsible, before being excised, for the sorting of these APs to the protein storage vacuole (PSV) and to protein bodies, but when isolated it has the capacity to sort other proteins to the vacuole, being considered an unconventional vacuolar sorting domain [16].…”

“…The PSI is a unique domain, with approximately 100 amino acids, present in the primary structure of some plant aspartic proteinases (APs) [13,14], which plays a role in germination, senescence, organism defense and protein turnover [15] This sequence is responsible, before being excised, for the sorting of these APs to the protein storage vacuole (PSV) and to protein bodies, but when isolated it has the capacity to sort other proteins to the vacuole, being considered an unconventional vacuolar sorting domain [16]. When associated with proteins, PSI can also determine their intracellular pathway depending on PSI's glycosylation status, in other words, PSI mediate a conventional pathway when glycosylated, but will follow a route, directly from the ER to the Golgi, when is not glycosylated [12]. This process is generically indicated as unconventional trafficking, and several examples have been described [10,[17][18][19].…”