Early sorting of inner nuclear membrane proteins is conserved

Abstract

Spodoptera frugiperda (Sf9) importin-alpha-16 is a translocon-associated protein that participates in the early sorting pathway of baculovirus integral membrane proteins destined for the inner nuclear membrane (INM). To discern whether sorting intermediate protein complexes like those observed in insect cells are also formed with mammalian INM proteins, cross-linked complexes of importin-alpha-16 with human lamin B receptor (LBR) and nurim were examined. Both LBR and nurim cross-link with Sf9 importin-alpha-16 during cotranslational membrane integration and remain proximal with importin-alpha-16 after integration into the endoplasmic reticulum membrane and release from the translocon. Human cells encode several isoforms of importin-alpha; to determine whether any of these isoforms may recognize INM-directed proteins, they were tested for their ability to cross-link with the viral-derived INM sorting motif sequence. One cross-linked adduct was detected with a 16-kDa isoform encoded from KPNA4 (KPNA-4-16). KPNA-4-16 was easily detected in microsomal membranes prepared from KPNA4-16 recombinant virus-infected cells and was also detected in microsomes prepared from HeLa cells. Together these observations suggest that elements of the early sorting pathway of INM-directed proteins mediated by importin-alpha-16 are highly conserved, and mammalian KPNA-4-16 is a candidate partner in sorting integral membrane proteins to the INM.

Fig. 1.

Cellular localization of LBR_1–238GFP transiently expressed in Sf9 cells. (a) Confocal microscopy images of LBR_1–238GFP. A single z-section is shown. Calnexin labeling is red, LBR_1–238GFP is green, and lamin Dm0 is white. For ease of viewing, lamin Dm0 is recolored in blue in the merge image. (b and c) Sf9 cells transiently expressing LBR_1-238GFP were fixed, thin sectioned, and analyzed by using GFP antibody and 25 nM gold-conjugated secondary antibody. The membranes are outlined, ONM and INM are labeled, and arrows point to the location of LBR_1–328GFP. (d) FRAP was used to determine recovery kinetics of LBR_1–238GFP in CHOK1 and Sf9 cells. (Upper) A graphical recovery curve of LBR_1–138GFP in Sf9 cells. (Lower) Calculated diffusion constants for the complete analyses. Total sample size is listed in parentheses.

Fig. 2.

LBR amino acid 203 is proximal to Sf9 importin-α-16. (a) The lysines in LBR_1–238 were changed to arginine (SI Fig. 6b) and then fused to a lysine-free sequence. The lysines available as cross-linking substrates are highlighted in red, transmembrane sequences are highlighted in yellow, and the lysine-free cassette containing the T7 epitope is highlighted in blue. (b–d) Schematics illustrate experimental protocol used in e. The protein marked with the red star is radiolabeled in the assay. b corresponds to the protocol used to generate data shown in lanes 1–4 and 8–10, c was used for lane 5, and d was used for lanes 6 and 7. (e) Radiolabeled LBR-KΔK was translated in the presence of microsomal membranes containing Sf9 importin-α-16-T7. The cross-linked adduct enriched on TALON beads is shown in lane 2 (∗) while lane 3 shows the adduct precipitated by using T7 antibody (∗). Lane 1 shows TALON-enriched product after treatment with buffer alone. The data presented in lanes 1–4 are from the same exposure of a single gel. To ensure that negative data shown in lane 1 are not due to a decreased quantity of enriched LBR-KΔK, the gel was exposed for a longer period, and the cross-linked adduct was not detected (data not shown). Lane 4 shows that the cross-linked adduct was not precipitated by using normal mouse IgG. Lane 5 shows the results of translation and cross-linking of truncated LBR-KΔK, and the cross-linked adduct is indicated (∗). In lanes 6 and 7, radiolabeled Sf9 importin-α-16-T7 was translated in the presence of membranes containing LBR-KΔK, and the cross-linked adduct enriched on TALON beads is noted (∗). In lanes 8–10, radiolabeled LBR-ΔK was translated in the presence of membranes containing Sf9 importin-α-16-T7 and exposed to buffer alone (lane 8); 50 or 100 μM BS³ (lanes 9 and 10) and the membrane pellet were analyzed. The arrow indicates the molecular mass of the expected cross-linked complex.

Fig. 3.

Nurim is proximal to Sf9 importin-α-16 both during and after cotranslational membrane integration. (a and b) Schematic illustrating the experimental design used for the data presented in c. Nurim is radiolabeled in these assays, and this is noted in the schematic with a red star. (c) Full-length (lanes 1–4) or truncated nurim (lanes 5–7) were translated in the presence of microsomal membranes containing Sf9 importin-α-16-T7 and exposed to buffer alone (lanes 1 and 5) or two concentrations of BS³ (noted below gel). The reaction was either treated with TALON beads (lanes 1–3, 5–7) or precipitated by using T7 antibody (lane 4). The exposure was increased for the T7 antibody precipitated lane. The cross-linked adduct is noted (∗).

Fig. 4.

KPNA-4–16 cross-links with INM-SM and is present in microsomal membranes. (a) KPNA-4–16 was translated in the presence of microsomal membranes containing the viral INM-SM cassette, treated with buffer or 200 μM BS³, and membrane pellet-resolved by using SDS/PAGE. The cross-linked sample is noted (∗). (b) Recombinant virus-infected cell extracts (30 μg per lane) were analyzed (KPNA-4–26-T7, lane 1; KPNA-4–16-T7, lane 2). A KPNA-4–16 isoform was detected when the slightly larger KPNA-4–26 gene was expressed. Microsomal membranes were prepared from KPNA-4–16-T7-infected cells, and KPNA-4–16 was easily detected by using T7 antibody (lane 3; 30-sec exposure; 30-eq microsomal membranes); however, two forms were detected. When a matched sample probed with KPNA-4-specific antibody was analyzed (compare lanes 3 and 4), substantially less positive product was detected (lane 4; 5-min exposure). KPNA4 antibody detected KPNA-4–16 in microsomal membranes prepared from HeLa cells (lane 5; 15-min exposure; 30-eq microsomal membranes), whereas no cross-reactivity was detected in microsomes prepared from Sf9 or wild-type baculovirus-infected cells (data not shown). KPNA-4–16 was not detected when analyzing total cell extracts (data not shown) or extracts of enriched nuclei (120 μg; lane 6).

Fig. 5.

Integrated model of directed trafficking of INM proteins. (a) Membrane associated importin-α-16 (KNPA-4–16) resides proximal to Sec61α. (b) During cotranslational membrane integration, INM-directed proteins are proximal to importin-α-16. (c) Importin-α-16 remains with the INM-directed proteins after ER membrane integration. INM-directed proteins can also associate with importin-α-16 after membrane integration. Thus, the class of mature protein does not appear to matter relative to INM protein-importin-α-16 interaction (Inset). (d) INM-directed proteins concentrate in membranes closely associated with the nucleus. (e) NLS sequences of INM-directed proteins form complexes with importins-α and -β and are translocated across the nuclear pore. (f and g) INM protein complexes disassemble (f) and interact (g) with their nucleoplasmic ligands.