The N-terminal region of Jaw1 has a role to inhibit the formation of organized smooth endoplasmic reticulum as an intrinsically disordered region

Abstract

Jaw1/LRMP is a type II integral membrane protein that is localized at the endoplasmic reticulum (ER) and outer nuclear membrane. We previously reported that a function of Jaw1 is to maintain the nuclear shape as a KASH protein via its carboxyl terminal region, a component of linker of nucleoskeleton and cytoskeleton complex in the oligomeric state. Although the oligomerization of some KASH proteins via the cytosolic regions serves to stabilize protein-protein interactions, the issue of how the oligomerization of Jaw1 is regulated is not completely understood. Therefore, we focused on three distinct regions on the cytosolic face of Jaw1: the N-terminal region, the coiled-coil domain and the stem region, in terms of oligomerization. A co-immunoprecipitation assay showed that its coiled-coil domain is a candidate for the oligomerization site. Furthermore, our data indicated that the N-terminal region prevents the aberrant oligomerization of Jaw1 as an intrinsically disordered region (IDR). Importantly, the ectopic expression of an N-terminal region deleted mutant caused the formation of organized smooth ER (OSER), structures such as nuclear karmellae and whorls, in B16F10 cells. Furthermore, this OSER interfered with the localization of the oligomer and interactors such as the type III inositol 1,4,5-triphosphate receptor (IP3R3) and SUN2. In summary, the N-terminal region of Jaw1 inhibits the formation of OSER as an IDR to maintain the homeostatic localization of interactors on the ER membrane.

Jaw1/LRMP increases Ca2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

“…For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1 (+) Hu SJaw1 (previously described 35 ) was digested with Bam HI and Xho I, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit (TaKaRa, #6023). For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described 35 ) was first performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil.…”

“…For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1 (+) Hu SJaw1 (previously described 35 ) was digested with Bam HI and Xho I, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit (TaKaRa, #6023). For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described 35 ) was first performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil. pcDNA3.1 (+) Hu LJaw1 Δcoil was then digested with Bam HI and Xho I, and the fragment was subcloned into a pcDNA5 FRT/TO vector (digested with the same enzymes) using the aforementioned DNA ligation kit, resulting in pcDNA5 FRT/TO Hu LJaw1 Δcoil.…”

Cleavage of the Jaw1 C-terminal region enhances its augmentative effect on the Ca2+release via inositol 1,4,5-trisphosphate receptors

“…3F). The NIDR, a structural flexible region previously described in our study (Kozono et al ., 2021), was fused between the C-terminal region of Jaw1 and the PA tag to increase the molecular mass of Fragment 2 on the electrophoresis gel. As shown in Figure 3G, Fragment 2 immunoprecipitated by anti-PA beads was specifically detected by SDS-PAGE followed by silver staining (closed black triangle), which was not detected in the lane of Ms Jaw1 PA as a control.…”

Jaw1/LRMP increases Ca2+ influx upon GPCR stimulation with heterogeneous effect on the activity of each ITPR subtype

“…For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1(+) Hu SJaw1 (previously described [35]) was digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit (TaKaRa, #6023). For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described [35]) was rst performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil.…”

“…For the production of pcDNA5 FRT/TO Hu SJaw1, pcDNA3.1(+) Hu SJaw1 (previously described [35]) was digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector digested with the same enzymes using a commercial DNA ligation kit (TaKaRa, #6023). For the production of pcDNA5 FRT/TO Hu SJaw1 Δcoil, pcDNA3.1 (+) Hu LJaw1 (previously described [35]) was rst performed by inverse PCR with primer set 1 resulting in pcDNA3.1 (+) Hu LJaw1 Δcoil. pcDNA3.1 (+) Hu LJaw1 Δcoil was then digested with BamHI and XhoI, and the fragment was subcloned into a pcDNA5 FRT/TO vector (digested with the same enzymes) using the aforementioned DNA ligation kit, resulting in pcDNA5 FRT/TO Hu LJaw1 Δcoil.…”