An evolutionarily conserved bimodular domain anchors ZC3HC1 and its yeast homologue Pml39p to the nuclear basket

Abstract

The proteins ZC3HC1 and TPR are structural components of the nuclear basket (NB), a fibrillar structure attached to the nucleoplasmic side of the nuclear pore complex (NPC). ZC3HC1 initially binds to the NB in a TPR-dependent manner and can subsequently recruit additional TPR polypeptides to this structure. Here, we examined the molecular properties of ZC3HC1 that enable its initial binding to the NB and TPR. We report the identification and definition of a nuclear basket-interaction domain (NuBaID) of HsZC3HC1 that comprises two similarly built modules, both essential for binding the NB-resident TPR. We show that such a bimodular construction is evolutionarily conserved, which we further investigated in Dictyostelium discoideum and Saccharomyces cerevisiae. Presenting ScPml39p as the ZC3HC1 homolog in budding yeast, we show that the bimodular NuBaID of Pml39p is essential for binding to the yeast NB and its TPR homologs ScMlp1p and ScMlp2p, and we further demonstrate that Pml39p enables linkage between subpopulations of Mlp1p. We eventually delineate the common NuBaID of the human, amoebic, and yeast homolog as the defining structural entity of a unique protein not found in all but likely present in most taxa of the eukaryotic realm.

Nuclear basket proteins regulate the distribution and mobility of nuclear pore complexes in budding yeast

“…of the copy number of Mlp1 (Supplemental Figure S2B). Pml39 was recently shown to anchor a subpopulation of Mlp1 to the NPC (Gunkel et al, 2023). To test whether Pml39 is required for the assembly of stable nuclear basket filaments, we performed doRITE with Nup133 and Mlp1 in pml39Δ cells.…”

“…The interpretation of Mlp1 as a stable Nup is corroborated by the observation that - once assembled - it is maintained at the NPC independent of Nup60 (Figure 2C), as also reported for meiotic budding yeast and mammalian cells (Aksenova et al, 2020; King et al, 2023). Interestingly, Mlp1 also forms stable assemblies at the NPC when PML39 is deleted (Figure 2C), suggesting that the additional copies of Mlp1 recruited to the NPC by Pml39 (Gunkel et al, 2023) are not required for assembly of stable nuclear basket filaments. Although we present clear evidence for the stable assembly formed by Mlp1, Mlp2 and Pml39 on the nucleoplasmic face of the NPC, we cannot exclude that a second more mobile pool exists which might be associated with the NE more generally as suggested by labelling at NE sites away from the NPCs by immuno-gold (Strambio-de-Castillia et al, 1999).…”

“…Nup60 recruits both Mlps as well as Nup2 to the nuclear basket via short binding sequences (Cibulka et al, 2022; Dilworth et al, 2001). Pml39 binds Mlp1 and to a lesser extend of Mlp1 to the NPC (Gunkel et al, 2023).…”

“…They, along with Nup2, are recruited to the NPC by Nup60 via short linear interacting motifs (Cibulka et al, 2022;Dilworth et al, 2001). Pml39 binds to Mlp1 and to a lesser extent to Mlp2 and promotes the binding of additional copies of Mlp1 to the NPC (Gunkel et al, 2023). While Nup1, Nup60 and Nup2 are found on all NPCs, the nuclear basket filaments are present only on a subset (Bensidoun et al, 2022;Galy et al, 2004;Palancade et al, 2005).…”

“…They, along with Nup2, are recruited to the NPC by Nup60 via short linear interacting motifs (Cibulka, Bisaccia, Radisavljević, Carrillo, & Köhler, 2022; Dilworth et al, 2001). Pml39 binds to Mlp1 and to a lesser extent to Mlp2 and promotes the binding of additional copies of Mlp1 to the NPC (Gunkel, Iino, Krull, & Cordes, 2023).…”