Surf4 modulates STIM1-dependent calcium entry

https://doi.org/10.1016/j.bbrc.2012.05.037Get rights and content

Abstract

Store-operated Ca2+ entry (SOCE) is crucial for various physiological responses in immune cells. Although it is known that STIM1 relocates into discrete puncta juxtaposed to the plasma membrane to initiate SOCE, the machinery modulating the function of STIM1 remains unclear. We explored to find its modulators using affinity purification for STIM1-binding proteins and identified surfeit locus protein 4 (Surf4). Surf4 associated with STIM1 in the endoplasmic reticulum. Deletion of Surf4 in DT40 B cells resulted in marked increase of SOCE and facilitation of STIM1 clustering upon store-depletion. These findings suggest the modulatory function of Surf4 for STIM1-mediated SOCE.

Highlights

► Surf4 was identified as a STIM1 binding partner. ► Surf4 interacts with STIM1 in the lumina of the ER. ► Surf4-deficeint DT40 B cells show a marked increase of SOCE. ► Absence of Surf4 facilitates STIM1 clustering after store-depletion.

Introduction

Store-operated Ca2+ entry (SOCE) through Ca2+ release-activated Ca2+ (CRAC) channel in immune cells is essential for gene regulation, cell proliferation, cytokine production and a variety of physiological responses [1], [2], [3], [4], [5]. Previous studies have identified STIM1, a Ca2+-binding membrane protein localized in the endoplasmic reticulum (ER), as the ER Ca2+ sensor and critical CRAC activator [6], [7]. STIM1 contains a Ca2+-binding EF-hand motif and a sterile alpha-motif (SAM) domain in the luminal side of the ER, and beyond the single transmembrane (TM) segment, it has two coiled-coil (CC) domains and carboxy-terminal cytoplasmic region including multiple serine and proline residues (S/P rich) [8]. At resting state, STIM1 distributes widely in the ER while the reduction of Ca2+ concentration in the ER induces dissociation of Ca2+ from EF-hand motif in STIM1 and subsequent oligomerization of STIM1 through EF-SAM domain [6]. This oligomerization leads to the relocalization of STIM1 into puncta at the ER–plasma membrane (PM) junction region [5], [9], which is absolutely required for the activation of SOCE [5], [8]. Subsequent studies have identified Orai1 (also known as CRACM1) as a pore subunit of the CRAC channels [10], [11]. The interaction of STIM1 with Orai1 is essential for gating of Orai1 to induce SOCE [5], [8], [12], [13]. In addition to Orai1, STIM1 has been reported to interact with several proteins such as STIM2 [14], sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) [15], end binding protein (EB1) [16], CRAC regulator 2A (CRACR2A) [17], voltage-gated Ca2+ channel CaV1.2 [18], [19], calnexin, exportin1, transportin1 [20], and ERp57 [21]. However, the molecular machinery modulating STIM1 activity remains poorly understood.

Here, we report that surfeit locus protein 4 (Surf4), the mammalian ortholog of the yeast cargo receptor Erv29p, is identified as a binding partner of STIM1, which modulates STIM1-mediated SOCE. Surf4 interacted with STIM1 in the lumina of the ER. Surf4-deficeint DT40 B cells showed a significant increase of SOCE in response to ER store deletion induced by the stimulation with B cell receptor (BCR) or thagsigargin (TG). Furthermore, in the absence of Surf4, clustering of STIM1 at the ER–PM junctions was markedly enhanced after BCR stimulation, suggesting that Surf4 regulates the clustering and/or relocation of STIM1 at the ER–PM junction area through their physical interaction. These observations provide the evidence for the modulatory function of Surf4 in STIM1-mediated SOCE.

Section snippets

Cells, Abs, and reagents

Wild-type and various mutant DT40 B cells were maintained in RPMI 1640 medium (Wako) supplemented with 10% FCS, 1% chicken serum, 50 mM 2-mercaptoethanol, 4 mM l-glutamate, and antibiotics. HeLa and 293T cells were cultured in DMEM (Wako) supplemented with 10% FCS and antibiotics. Anti-Flag mAb (M2) and anti-V5 Ab were purchased from Sigma or Invitrogen, respectively.

Crosslinking and affinity protein purification

Wild-type or Flag-STIM1 expressing STIM1-deficient DT40 B cells [22] were crosslinked with 1% w/v paraformaldehyde (PFA) for 5 min

Identification of STIM1 binding partners

To identify novel regulators of SOCE, affinity purification for STIM1 was conducted by using DT40 B cells stably expressing Flag-STIM1. We used in vivo PFA cross-linking of cells to preserve STIM1 protein complexes and to validate possible STIM1-interacting proteins. Formaldehyde is a highly specific cross-linker that is reactive with primary amines to one another, is easily reversible, and is used to preserve protein–protein complexes [25]. To identify the binding proteins of Flag-STIM1, the

Acknowledgments

We thank R. Tsien (University of California at San Diego) for providing the pRSETB-mCherry plasmid. We are thankful to M. Matsumoto for critical reading of the manuscript. This work was supported by Grant-in-Aids for the Ministry of Education, Culture, Sports, Science and Technology, Japan (Y.B. and T.K.) and for Japan Science and Technology Agency and Core Research for Evolutional Science and Technology (T.K.) and “HISHO” The Top Thirty Young Researchers of Osaka University (Y.B.).

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