Sec16B is involved in the endoplasmic reticulum export of the peroxisomal membrane biogenesis factor peroxin 16 (Pex16) in mammalian cells

Abstract

Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.

Fig. 1.

Identification of Sec16B and the specificity of siRNAs. (A) Lysates (30 μg) of 293T cells treated with lamin A/C siRNA (lane 1), Sec16B siRNA-1 (lane 2), or Sec16B siRNA-2 (lane 3) were subjected to SDS/PAGE and then analyzed by Western blotting with an anti-Sec16B antibody. The asterisk and double asterisk denote protein bands nonspecifically labeled by the anti-Sec16B antibody. (B) HeLa cells stably expressing GFP-Sec16B were treated with lamin A/C siRNA (Top), Sec16B siRNA-1 (Middle), or Sec16B siRNA-2 (Bottom) and stained with Hoechst 33342. (Scale bar, 10 μm.) (C) HeLa cells stably expressing GFP-Sec16B were treated with lamin A/C siRNA (lane 1), Sec16A siRNA (lane 2), Sec16B siRNA-1 (lane 3), or Sec16B siRNA-2 (lane 4). Lysates (30 μg) of cells were subjected to SDS/PAGE and analysis by Western blotting.

Fig. 2.

Sec16B is tightly associated with ER membranes. Subcellular fractionation was performed as described in Materials and Methods. The proteins (30 μg) in each fraction were resolved by SDS/PAGE and then analyzed by Western blotting. C, cytosol; HM, heavy membrane fraction; M, microsomal fraction; PNS, postnuclear supernatant.

Fig. 3.

Overexpression of Sec16B causes redistribution of Pex3-GFP and Pex16-GFP to the ER. (A) HeLa cells were transfected with the plasmid encoding FLAG-Sec16B (Upper) or FLAG-Sec16A (Lower). At 24 h after transfection, the cells were double-stained with antibodies against FLAG and catalase. (Scale bar, 10 μm.) (B) Quantitation of the data shown in A. Data are for three independent experiments and represent the means ± SD. (C and D) HeLa cells stably expressing Pex3-GFP (Upper) or Pex16-GFP (Lower) were transfected with the plasmid encoding FLAG-Sec16B (C) or FLAG-Sec16A (D), and stained with antibodies against FLAG and calnexin. (Scale bars, 10 μm.) The asterisks indicate cells overexpressing FLAG-Sec16B or FLAG-Sec16A.

Fig. 4.

Knockdown of Sec16B induces elongation of peroxisomes. HeLa cells were treated with lamin A/C siRNA (Top), Sec16A siRNA (Upper Middle), Sec16B siRNA-1 (Lower Middle), or Sec16B siRNA-2 (Bottom) and stained with an antibody against catalase (A) or PMP70 (B). Images (Right) show higher magnification views of the boxed regions (Left). (Scale bars, 10 μm.) (C) Quantitation of the data shown in B. Data are for three independent experiments and represent the means ± SD.

Fig. 5.

Knockdown of Sec16B induces elongation of peroxisomes and reduces their number. HeLa cells were mock-treated (A) or treated with Sec16B siRNA-1 for 72 h (B), fixed with 4% (wt/vol) paraformaldehyde and 0.1% glutaraldehyde for 30 min, and processed for immunoelectron microscopy. The arrows indicate peroxisomes visualized with anti-PMP70. (Scale bar, 500 nm.)

Fig. 6.

Knockdown of Sec16B causes redistribution of Pex16-GFP and degradation of Pex3. HeLa cells stably expressing Pex16-GFP (A) or Pex3-GFP (B) were treated with lamin A/C siRNA (Top), Sec16A siRNA (Upper Middle), Sec16B siRNA-1 (Lower Middle), or Sec16B siRNA-2 (Bottom) and stained with an antibody against calnexin (A) or catalase (B). (Scale bars, 10 μm.) (C) HeLa cells stably expressing Pex3-GFP were mock-treated (lanes 1–4) or treated with Sec16B siRNA-1 (lanes 5–8), lysed, and then subjected to subcellular fractionation. The proteins (30 μg) in each fraction were resolved by SDS/PAGE and then analyzed by Western blotting. C, cytosol; HM, heavy membrane fraction; M, microsomal fraction; PNS, postnuclear supernatant. (D) HeLa cells were transfected with lamin A/C siRNA, Sec16A siRNA, Sec16B siRNA-1, or Sec16B siRNA-2. At 72 h after transfection, cells were lysed immediately (0 h) or after incubation with 10 μg/mL MG132 for 6 h and then subjected to SDS/PAGE and analysis by Western blotting.

Fig. 7.

Localization of Sec16B in ERESs is not relevant to its role in peroxisome biogenesis. (A) HeLa cells were treated with Sec16B siRNA-1 for 48 h and then transfected with the plasmids encoding GFP-Sec16B fragments resistant to Sec16B siRNA-1. At 24 h after transfection with the plasmid, the cells were fixed and stained with an antibody against catalase. (Scale bar, 10 μm.) (B) Quantitation of the data shown in A. Data are for three independent experiments and represent the means ± SD.