Sec16B is involved in the endoplasmic reticulum export of the peroxisomal membrane biogenesis factor peroxin 16 (Pex16) in mammalian cells Shusuke Yonekawaa, Akiko Furunoa, Takashi Babaa, Yukio Fujikib,c, Yuta Ogasawarad, Akitsugu Yamamotod, Mitsuo Tagayaa, and Katsuko Tania,1 aSchool of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan; bDepartment of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan; cCore Research of Evolutional Science and Technology, Japan Science and Technology Agency, Chiyoda-ku, Tokyo 102-0075, Japan; and dFaculty of Bioscience, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan Edited by Randy Schekman, University of California, Berkeley, CA, and approved June 20, 2011 (received for review March 1, 2011) Sec16 plays a key role in the formation of coat protein II vesicles, from the endoplasmic reticulum (ER) en route to peroxisomes which mediate protein transport from the endoplasmic reticulum (13). In addition, several lines of evidence suggest that the ER (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: participates in the de novo formation of peroxisomes (13–20). A Sec16A, which is a longer primary ortholog of yeast Sec16, and very recent study involving a yeast cell-free system revealed Sec16B, which is a shorter distant ortholog. Previous studies have that ER-peroxisome carriers are formed in a Pex19-dependent shown that Sec16B, as well as Sec16A, defines ER exit sites, where manner (21). coat protein II vesicles are formed in mammalian cells. Here, we In this report, we show that Sec16B plays an important role in reveal an unexpected role of Sec16B in the biogenesis of mamma- the transport of Pex16 from the ER to peroxisomes in mam- lian peroxisomes. When overexpressed, Sec16B was targeted to malian cells. Sec16 was first characterized in yeast S. cerevisiae as a the entire ER, whereas Sec16A was mostly cytosolic. Concomitant 240-kDa peripheral membrane protein that interacts with coat with the overexpression of Sec16B, peroxisomal membrane bio- protein II (COPII) coat components and facilitates their assembly genesis factors peroxin 3 (Pex3) and Pex16 were redistributed from and vesicle budding (22–25). In yeast Pichia pastoris, Sec16 defines peroxisomes to Sec16B-positive ER membranes. Knockdown of ER exit sites (ERESs) (26), special domains where COPII-coated Sec16B but not Sec16A by RNAi affected the morphology of vesicles are formed (27). There are two mammalian orthologs, peroxisomes, inhibited the transport of Pex16 from the ER to per- Sec16A (250 kDa) and Sec16B (117 kDa) (also referred to as oxisomes, and suppressed expression of Pex3. These phenotypes Sec16L and Sec16S, respectively) (28–30). Sec16A, which is lo- fi were signi cantly reversed by the expression of RNAi-resistant calized in cup-like structures in ERESs (31), appears to be the Sec16B. Together, our results support the view that peroxisomes primary Sec16 ortholog because its molecular mass is similar to are formed, at least partly, from the ER and identify a factor re- that of Sec16 in yeast (22) and Drosophila (32). Sec16B, which sponsible for this process. appears to be conserved in vertebrates, is also localized in ERESs, but its function has not been fully examined in the context of ost eukaryotic cells contain peroxisomes, which are single membrane trafficking. Our results suggest that Sec16B may par- Mmembrane-bound organelles that function in various met- ticipate in the formation of new peroxisomes derived from the ER. abolic pathways, including the β-oxidation of fatty acids, bio- synthesis of plasmalogens and bile acids, and hydrogen peroxide Results metabolism (1). To perform this variety of functions, perox- Sec16B Is Tightly Associated with ER Membranes. To characterize isomes are highly dynamic; their number, size, and function fi change in response to cellular conditions. In addition, unlike Sec16B, we rst produced a polyclonal anti-Sec16B antibody. mitochondria, peroxisomes can be formed through de novo The antibody reacted with a 120-kDa band on Western blots of synthesis as well as through the growth and division of preex- 293T cell lysates, and the intensity of the band markedly de- isting peroxisomes (2, 3). creased when cells were treated with siRNAs targeting Sec16B Peroxisomal matrix proteins are synthesized on free ribosomes (siRNA-1 and siRNA-2) (Fig. 1A), suggesting that the 120-kDa in the cytosol and posttranslationally imported to peroxisomes band is Sec16B. The specificity of these siRNAs was confirmed (4). This import pathway includes the recognition of two distinct by the finding that they are able to knock down GFP-Sec16B peroxisomal targeting signals (PTS1 and PTS2) by peroxin 5 stably expressed in HeLa cells (Fig. 1 B and C). (Pex5) and Pex7, respectively, followed by translocation across We carried out subcellular fractionation and analyzed each the membrane through the import machinery, including Pex14 fraction by Western blotting with the above antibody (Fig. 2). and Really Interesting New Gene peroxins (5, 6). The import Sec16B was almost exclusively fractionated into the microsomal pathway for peroxisomal membrane proteins (PMPs), on the fraction (lane 3). Little Sec16B was detected in the heavy other hand, is believed to be independent of that used by matrix membrane fraction rich in peroxisomes (catalase) and mito- proteins. Genetic phenotype complementation analysis of yeast and mammalian mutants devoid of peroxisome membranes chondria (Tom 20) (lane 2) or in the cytosol (lane 4). It seemed revealed that Pex3, Pex16, and Pex19 are essential for PMP that Sec16B bound more tightly to membranes than Sec16A did. import (references in ref. 7). Pex3 is a PMP import receptor (8), and Pex19 is a chaperone and import receptor for most PMPs (9). Pex16 appears to function as a Pex3-Pex19 receptor in Author contributions: S.Y., M.T., and K.T. designed research; S.Y., Y.O., and A.Y. per- mammals (7) and as a negative regulator of peroxisome fission in formed research; Y.F. contributed new reagents/analytic tools; A.F., T.B., Y.O., and A.Y. yeast Yarrowia lipolytica (10) but is absent in Saccharomyces analyzed data; and M.T. and K.T. wrote the paper. cerevisiae (11). The authors declare no conflict of interest. Although compelling evidence suggests that PMPs are trans- This article is a PNAS Direct Submission. ported directly from the cytosol to peroxisomes (7–9, 12), recent 1To whom correspondence should be addressed. E-mail: [email protected]. work has suggested that some PMPs, including the PMP import This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. receptors Pex3 and Pex16, seem to be, at least partly, transported 1073/pnas.1103283108/-/DCSupplemental. 12746–12751 | PNAS | August 2, 2011 | vol. 108 | no. 31 www.pnas.org/cgi/doi/10.1073/pnas.1103283108 Downloaded by guest on September 24, 2021 data in Fig. 3B). These results raised the possibility that Sec16B is involved in the biogenesis of peroxisomes. Recent studies suggested that peroxisomes can arise de novo from the ER not only in yeast and plant cells (14–19) but in mammalian cells (13, 20). In mammalian cells, two PMPs, Pex3 and Pex16, have been proposed to regulate this ER-derived pathway (13, 20). To explore the possibility that Sec16B is in- volved in this process, we examined the distribution of Pex3-GFP and Pex16-GFP in Sec16B-overexpressing cells. As shown in Fig. 3C, when Sec16B was overexpressed, both Pex3-GFP (Fig. 3C, Upper) and Pex16-GFP (Fig. 3C, Lower) were redistributed, with the punctate pattern changing into a perinuclear aggregated distribution. Concomitantly, the calnexin staining changed from a reticular pattern to an aggregated pattern surrounding the nucleus, although calnexin and Pex16p-GFP were not completely colocalized. In contrast, overexpression of Sec16A did not Fig. 1. Identification of Sec16B and the specificity of siRNAs. (A) Lysates (30 markedly affect the localization of Pex3-GFP or Pex16-GFP, and μg) of 293T cells treated with lamin A/C siRNA (lane 1), Sec16B siRNA-1 (lane 2), overexpressed Sec16A was mostly cytosolic (Fig. 3D). 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 Sec16B Depletion Affects Peroxisome Morphology and the Distribution asterisk denote protein bands nonspecifically labeled by the anti-Sec16B of PMPs. To elucidate the involvement of Sec16B in the bio- antibody. (B) HeLa cells stably expressing GFP-Sec16B were treated with lamin genesis of peroxisomes, cells were treated with siRNA targeting A/C siRNA (Top), Sec16B siRNA-1 (Middle), or Sec16B siRNA-2 (Bottom) and Sec16B and then analyzed by immunofluorescence microscopy. stained with Hoechst 33342. (Scale bar, 10 μm.) (C) HeLa cells stably expressing As shown in Fig. 4A, elongated catalase-positive puncta were GFP-Sec16B were treated with lamin A/C siRNA (lane 1), Sec16A siRNA (lane 2), μ seen in cells depleted of Sec16B by Sec16B siRNA-1 (Fig. 4A, Sec16B siRNA-1 (lane 3), or Sec16B siRNA-2 (lane 4). Lysates (30 g) of cells were Lower Middle), whereas no significant change was observed in subjected to SDS/PAGE and analysis by Western blotting. cells treated with siRNA targeting Sec16A (Fig. 4A, Upper Mid- dle). Elongated catalase-positive staining structures were also Overexpression of Sec16B Causes the Redistribution of Pex3-GFP and observed in cells treated with Sec16B siRNA-2 (Fig. 4A, Bottom). A similar pattern was observed on staining for PMP70 (Fig. 4 B Pex16-GFP to the ER. To compare the function of Sec16B with that and C). The results of immunoelectron microscopic analysis (Fig. of Sec16A, each protein with a FLAG tag was overexpressed, fi fl 5) con rmed that the elongation of peroxisomes occurred fol- and organelle morphology was then examined by immuno uo- lowing Sec16B knockdown.