A conserved membrane-binding domain targets proteins to organelle contact sites

Abstract

Membrane contact sites (MCSs), where the membranes of two organelles are closely apposed, are regions where small molecules such as lipids or calcium are exchanged between organelles. We have identified a conserved membrane-binding domain found exclusively in proteins at MCSs in Saccharomyces cerevisiae. The synaptotagmin-like-mitochondrial-lipid binding protein (SMP) domain is conserved across species. We show that all seven proteins that contain this domain in yeast localize to one of three MCSs. Human proteins with SMP domains also localize to MCSs when expressed in yeast. The SMP domain binds membranes and is necessary for protein targeting to MCSs. Proteins containing this domain could be involved in lipid metabolism. This is the first protein domain found exclusively in proteins at MCSs.

Fig. 1.

Diagrams of yeast proteins containing SMP domains. Nterm, N-terminus; Cterm, C-terminus; other abbreviations are as given in the text.

Fig. 2.

Nvj2p is localized to NVJ and is not required for NVJ formation. (A) Localization of Nvj2–GFP under the GALl1 promoter expressed for 2 hours in a strain (ATY182) with RFP–HDEL as a marker of the ER. (B) Cell expressing Nvj2–GFP and the NVJ marker Nvj1–mCherry (strain ATY192). Nvj2–GFP expression was induced 2 hours before examination. (C) Nvj2–GFP under the GALl1 promoter was expressed for 2 hours in wild-type, nvj1Δ and vac8Δ yeast strains (ATY192, ATY180 and ATY181, respectively). (D) Examples of cells with weak and strong localization of Nvj2–GFP at the NVJ. Cells with Nvj2–GFP localized to PMN blebs and vesicles are also shown. (E) Growth curve (measured as the OD at 600 nm) of cells (strain ATY104) expressing Nvj2–GFP and cultured in YPD medium. (F) Percentage of cells showing fluorescence at the NVJ and in PMN structures at different time points of the growth curve in E. (G) GFP–OSH1, which localizes to the NVJ, expressed in wild-type, nvj1Δ and nvj2Δ strains (ATY214, ATY183 and ATY187, respectively). Scale bars: 5 μm.

Fig. 3.

The tricalbins localize to ER–PM contacts. (A) Cells expressing Tcb1–GFP, Tcb2–GFP or Tcb3–GFP and the ER marker RFP–HDEL (strains WPY971, WPY973 and WPY975, respectively). Images were taken focusing on the middle and periphery of the cells. (B) Localization of GFP fused to the indicated portions of Tcb2p. The fusion proteins, under the GALl1 promoter, were expressed for 2 hours in strains containing the ER marker RFP–HDEL (strains ATY33, ATY34, ATY36, ATY37, ATY38). Scale bars: 5 μm.

Fig. 4.

The cortical ER is closely apposed to the plasma membrane. (A) Diagram of the interaction between Sec63-F1 in the ER and Pma1-F2 in the PM. Sec63(1–250)-F1 contains the first 250 amino acids of Sec63p and lacks the C-terminal cytosolic domain of Sec63p. (B,C) Cells expressing the indicated fusion proteins and the ER marker RFP–HDEL (strains ATY57, ATY98, ATY97, ATY54). Arrowheads indicate peripheral ER that is not close to the PM and lacks Venus signal. The images in C were taken focusing on the middle and periphery of the cells. Scale bars: 5 μm.

Fig. 5.

Human orthologues of the tricalbins and Nvj2p localize to MCSs in yeast. (A) The human tricalbin homologue E-Syt1 was fused to GFP and expressed together with the ER marker RFP–HDEL (strain ATY196). (B) The human Nvj2p homologue HT008 was fused to GFP and expressed with the NVJ marker Nvj1–mCherry (strain ATY195). Scale bars: 5 μm.

Fig. 6.

The SMP domain is necessary for MCS localization. (A) Cells expressing either full-length Tcb2–GFP (left panels) or the protein lacking the SMP domain (right panels) and the ER marker RFP–HDEL (strains ATY33 and ATY84, respectively). (B) Cells expressing either full-length Nvj2–GFP (left panels) or the protein lacking the SMP domain (right panels) and the ER marker RFP–HDEL (strains ATY182 and ATY250, respectively). (C) Cells expressing either full-length Mmm1–GFP (left panels) or the protein lacking the SMP domain (right panels) and the ER marker RFP–HDEL (strains ATY140, ATY142). (D,E) Images of cells expressing the indicated fusion proteins were coexpressed with the ER marker ssRFP–HDEL, ERMES marker Mmm1–mCherry or the NVJ marker Nvj1–mCherry (strains ATY127, ATY257, ATY218 and ATY217, respectively). Note that the Tcb2p, Mmm1p and Nvj2p portions of the fusions lacked their N-termini and TMDs. Images were taken of cells in mid-logarithmic growth phase (D) or late-logarithmic growth phase (E). In all panels, expression of GFP fusion proteins was induced 2 hours before examination. Scale bars: 5 μm.

Fig. 7.

The SMP domain is a membrane-binding domain. (A) MBP fusions to the indicated SMP domains were expressed in yeast and purified on magnetic beads coated with anti-MBP antibody. The amount of protein bound to beads: 10 pmole MBP (in strain ATY287), 4 pmole MBP–Mdm12 (in strain ATY288), 6 pmole MBP–SMP(Nvj2p; in strain ATY290), and 0.2 pmole MBP–SMP(Tcb2p; in strain ATY289). The beads were mixed with liposomes with various lipid compositions and trace [³H]triolein for 30 minutes at 30°C (mol% of liposomes: ER-like, PC:PS:PE:PI, 50:10:30:10; PM-like, PC:PS:PE:PI:PtdIns(4,5)P₂:cholesterol, 12:28:20:20:1:20; mitochondria-like, PC:CL:PE:PI, 50:10:30:10; vacuole-like, PC:PS:PE:PI: PtdIns(3,5)P₂, 50:5:22.5:22.5:1). The beads were washed twice and counted in a scintillation counter. The counts per minute were normalized to that of the MBP sample. (B) Lysates from yeast strains expressing the indicated SMP domains fused to GFP were separated on Optiprep gradients as described in Materials and Methods. Five equal fractions were taken from the top (fraction 1) to the bottom (fraction 5) of the gradient and immunoblotted with antibodies against GFP and the PM proteins Pma1p (strains ATY1, ATY38, ATY153, ATY135). PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PtdIns(3,5)P₂, phosphatidylinositol (3,5)-bisphosphate; PtdIns(4,5)P₂, phosphatidylinositol (4,5)-bisphosphate; PS, phosphatidyl serine.

Fig. 8.

Sensitivity of cells missing SMP-containing proteins to lipid biosynthesis inhibitors. Cultures of the indicated strains were grown to mid-logarithmic growth phase in YPD and adjusted to OD_600nm=1. Serial tenfold dilutions were spotted on (A) SC plates containing the indicated concentrations of myriocin and (B) YPD plates with the indicated concentrations of fenpropimorph. The plates were incubated for 4 days at 30°C. (Strains BY4741, ATY109, ATY291, ATY292, ATY293, ATY294, WPY267 and CVY551).