The Intracellular Transport and Secretion of Calumenin- 1/2 in Living Cells Qiao Wang1., Hui Feng1., Pengli Zheng1., Birong Shen1, Liang Chen1, Lin Liu1, Xiao Liu1, Qingsong Hao1, Shunchang Wang1, Jianguo Chen1,2, Junlin Teng1* 1 State Key Laboratory of Bio-membrane and Membrane Bio-engineering and Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, College of Life Sciences, Peking University, Beijing, China, 2 Center for Theoretical Biology, Peking University, Beijing, China Abstract Calumenin isoforms 1 and 2 (calu-1/2), encoded by the CALU gene, belong to the CREC protein family. Calu-1/2 proteins are secreted into the extracellular space, but the secretory process and regulatory mechanism are largely unknown. Here, using a time-lapse imaging system, we visualized the intracellular transport and secretory process of calu-1/2-EGFP after their translocation into the ER lumen. Interestingly, we observed that an abundance of calu-1/2-EGFP accumulated in cellular processes before being released into the extracellular space, while only part of calu-1/2-EGFP proteins were secreted directly after attaching to the cell periphery. Moreover, we found the secretion of calu-1/2-EGFP required microtubule integrity, and that calu-1/2-EGFP-containing vesicles were transported by the motor proteins Kif5b and cytoplasmic dynein. Finally, we determined the export signal of calu-1/2-EGFP (amino acid positions 20–46) and provided evidence that the asparagine at site 131 was indispensable for calu-1/2-EGFP stabilization. Taken together, we provide a detailed picture of the intracellular transport of calu-1/2-EGFP, which facilitates our understanding of the secretory mechanism of calu-1/2. Citation: Wang Q, Feng H, Zheng P, Shen B, Chen L, et al. (2012) The Intracellular Transport and Secretion of Calumenin-1/2 in Living Cells. PLoS ONE 7(4): e35344. doi:10.1371/journal.pone.0035344 Editor: Sara Salinas, CNRS, France Received August 16, 2011; Accepted March 14, 2012; Published April 13, 2012 Copyright: ß 2012 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Major State Basic Research Development Program of China (973 Program)(2010CB833705) and the National Natural Science Foundation of China (30971433, 31071177). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] . These authors contributed equally to this work. Introduction Despite the initial characterization of calu-1/2, many aspects are still elusive, including their intracellular transport and Human calumenin (calu), a CREC protein family member, is secretory process. Here, we used calu-1/2-EGFP, in which the encoded by the CALU gene (NCBI GeneID: 813) [1,2], which is EGFP would possibly mask the C-terminal HDEF retention signal, mapped on chromosome 7q32 [3]. Two alternative spliced to study its secretion process. We reported the translocation of variants of the CALU gene are identified as calu-1 and calu-2 calu-1/2-EGFP into the lumen of the endoplasmic reticulum (ER), (also known as crocalbin) [4]. The two isoforms have equal lengths visualized their intracellular transport in the vesicles, and showed (315 amino acids), with exons 3 and 4 exchanged [5], and are the secretion of calu-1/2-EGFP through either ‘‘secretion after ubiquitously expressed in human tissues [6]. Both calu-1 and -2 attachment’’ or ‘‘secretion after accumulation’’. To investigate the contain an N-terminal signal sequence (19 amino acids) and seven 2+ underlying transport mechanism, we determined the roles of EF-hand domains for binding Ca [7]. Previous reports show that cytoskeleton network and motor proteins on the intracellular they localize to the secretory pathway and are secreted into the transport and secretion of calu-1/2-EGFP. Furthermore, we extracellular space [8–10], while some researches insist that calu- identified that Kif5b and cytoplasmic dynein were the motors 1/2 contained an ER-retaining signal HDEF at the C-terminus, that were responsible for their microtubule-dependent trafficking. and are retained in the ER lumen [11–13]. Besides, proline at the Finally, mutational analysis revealed the export signal of calu-1/2- +2 position from the predicted signal peptide cleavage site of calu- EGFP and amino acid point crucial for the calu-1/2-EGFP 1/2 [6] acts as an export signal to mediate calu-1/2 secretion [14]. stability. Extracellularly, calu-1 is reported to interact with the serum 2+ amyloid P component in the presence of Ca , indicating its Results possible role in amyloidosis [15]. Calu-1 may also be involved in autocrine and paracrine signaling since it decreases the expression Calu-1/2 are translocated into the ER lumen and secreted level of septin 2 and actin fragments as well as regulating the cell With our produced antibody, which recognize both calu-1 and cycle in fibroblasts [16]. Recently, thrombospondin-1, a secreted calu-2 isoforms (data not shown), we found that a great deal of glycoprotein, is reported to form a complex with calu-1, suggesting calu-1/2 were detected in the cultured medium (Figure 1A). that calu-1/2 play a potential role in hemostasis and thrombosis However, immunofluorescence assay showed that calu-1/2 [2]. localized throughout the cell in an ER-like manner rather than PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35344 Intracellular Transport of Calumenin accumulated at the Golgi apparatus both in HeLa and HEK293T whereas b4GalT-1-EGFP, whose EGFP is on the luminal side of cells (Figure S1). In order to specifically study the secretory process the ER and the Golgi apparatus, was not immunolabeled of calu-1/2, we used calu-1/2-EGFP, in which the C-terminal ER- (Figure 1E). Also, anti-EGFP antibody did not label the retaining signal HDEF was disrupted by the EGFP tag. A majority overexpressed calu-1/2-EGFP by immunofluorescence after of calu-1/2-EGFP fusion proteins were detected in the cultured digitonin treatment (Figure 1E), suggesting that calu-1/2 localized medium as expected (Figure 1B). Meanwhile, calu-1/2-EGFP in the lumen of the ER and the Golgi apparatus. Furthermore, to accumulated at the Golgi apparatus both in HeLa and HEK293T determine the signal responsible for the entry of calu-1/2 into the cells (Figure 1C and 1D), suggesting that the ER-retaining lumen, we generated several constructs of calu-2 and visualized pathway was blocked. their subcellular localization, and found that the 19 amino acids on We then used digitonin, which selectively permeabilizes the the N-terminal were necessary and sufficient for the Golgi plasma membrane but not the intracellular membranes [17], to localization of calu-2-EGFP (Figure 1F). These results, together treat the cells after fixation. Under this treatment, GM130, which with the previous report that the N-terminal 19 amino acids of peripherally attaches to the cytoplasmic surface of the Golgi calu-1 are cleaved [14], suggest that calu-1/2-EGFP are secreted apparatus, was detected by its antibody, whereas the ER luminal to the extracellular space, and that the N-terminal 19 amino acids protein PDI could not be recognized by its own antibody act as a signal peptide, which docks calu-1/2-EGFP to the ER and (Figure S2). Similarly, the cytosolic EGFP tag of APP-EGFP then translocates them into the lumen of the endomembrane fusion protein was immunolabeled by anti-EGFP antibody, system. Figure 1. Calu-1/2 are translocated into the lumen and secreted. (A) Western blotting analysis of endogenous calu-1/2 from total cell lysate (TCL) and cultured medium (CM) of HeLa cells. (B) Western blotting analysis of overexpressed calu-1/2-EGFP of HeLa cells. Samples of TCL and CM were separated by SDS-PAGE and probed with anti-EGFP and anti-tubulin antibodies. Coomassie Blue staining (Co.St.) was used as a loading control of CM. (C) Immunofluorescence of EGFP or Calu-1/2-EGFP overexpressing HeLa cells probed with anti-Giantin antibody. Bar, 10 mm. (D) Immunofluorescence of EGFP or Calu-2-EGFP overexpressing HEK293T cells probed with anti-Giantin antibody. Bar, 10 mm. (E) HeLa cells were immunofluorescently labeled by the indicated antibodies after being permeabilized by digitonin. Scale bar, 10 mm. (F) Subcellular localization of 11 EGFP-fusion proteins of calu-2 transcripts in HeLa cells, which were co-transfected with GRIP-mRFP and permeabilized by Triton X-100 after fixation. Numbers indicate the amino acid positions in calu-2. In the cartoon, transcripts which localized on the secretory pathway are marked by green ticks. Red crosses indicate the transcripts that are smeared in HeLa cells. Scale bar, 10 mm. doi:10.1371/journal.pone.0035344.g001 PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e35344 Intracellular Transport of Calumenin Intracellular transport of calu-1/2-EGFP Two types of calu-1/2-EGFP secretion To monitor the intracellular transport and secretory process of To further visualize the secretory process of calu-1/2-EGFP, we calu-1/2-EGFP, we monitored the distribution and dynamic observed in more detail the in vivo behavior of calu-1/2-EGFP at behavior of calu-1/2-EGFP in live HeLa cells ($30 cells) using an the periphery of the cells, and found two main types of secretion. Andor spinning disk confocal microscope system.