Mdm1/Snx13 is a novel ER-endolysosomal interorganelle tethering protein

Abstract

Although endolysosomal trafficking is well defined, how it is regulated and coordinates with cellular metabolism is unclear. To identify genes governing endolysosomal dynamics, we conducted a global fluorescence-based screen to reveal endomembrane effector genes. Screening implicated Phox (PX) domain-containing protein Mdm1 in endomembrane dynamics. Surprisingly, we demonstrate that Mdm1 is a novel interorganelle tethering protein that localizes to endoplasmic reticulum (ER)-vacuole/lysosome membrane contact sites (MCSs). We show that Mdm1 is ER anchored and contacts the vacuole surface in trans via its lipid-binding PX domain. Strikingly, overexpression of Mdm1 induced ER-vacuole hypertethering, underscoring its role as an interorganelle tether. We also show that Mdm1 and its paralogue Ydr179w-a (named Nvj3 in this study) localize to ER-vacuole MCSs independently of established tether Nvj1. Finally, we find that Mdm1 truncations analogous to neurological disease-associated SNX14 alleles fail to tether the ER and vacuole and perturb sphingolipid metabolism. Our work suggests that human Mdm1 homologues may play previously unappreciated roles in interorganelle communication and lipid metabolism.

Figure 1.

Mdm1 is a novel interorganelle tether. (A) Schematic of Mup1-pH trafficking screen. (B) Stages of Mup1-pH screen. (C) Table of several mutants detected in screening. Images are representative mdm1Δ Mup1-pH cells displaying class 2/3 sorting defects. (D) Domain architecture of Mdm1/Snx13 family proteins. (E) Yeast expressing Mdm1-GFP and DsRed-HDEL (ER) marker and stained with the vacuole marker CMAC (white). (F) Yeast colabeled with Mdm1-GFP and Nvj1-mCherry. A schematic of Mdm1-GFP at the NVJ. Dotted lines indicate the cell periphery, based on DIC images. MVB, multivesicular body; N, nucleus; V, vacuole. Bars, 5 µm.

Figure 2.

Mdm1 is anchored in the ER by its transmembrane region and binds in trans to vacuolar PI3P. (A–C) Yeast colabeled with DsRED-HDEL, CMAC, and either FL Mdm1 (A), Mdm1^1toPXA-GFP (B), or the Mdm1 PX domain only (Mdm1^PX-GFP; C). A linear profile of the cell shows Mdm1-GFP colocalization relative to the ER and/or vacuole. The linear profiles are profiles from single cells that are highly representative of many cells from at least three independent experiments, where >100 cells were observed to display an Mdm1-GFP distribution like this in each experiment. (D) Alignment of the lipid-binding region of PX domains, with R823 denoted. Structure of the PX domain of Snx14 (Protein Data Bank accession no. 4PQO; Mas et al., 2014). (E) Yeast stained with CMAC and expressing either GFP-Mdm1 PX (top) or the lipid-binding mutant GFP-Mdm1 PX R823E (bottom). (F and G) Yeast stained with CMAC (vacuole) and expressing either wild-type Mdm1 FL–GFP (F) or Mdm1 FL with R823E (G). Dotted lines indicate the cell periphery based on DIC images. AU, arbitrary units; N, nucleus; V, vacuole. Bars, 5 µm.

Figure 3.

Overexpression of Mdm1 hypertethers the ER and vacuole. (A) Yeast stained with CMAC (vacuole; white) and expressing DsRed-HDEL (ER; red) and Mdm1-GFP with either its endogenous promoter (top) or the overexpression GPD promoter (bottom). Bar, 5 µm. (B) Quantitation of NVJ length in >30 cells. Error bars represent the standard error of the measurements. (C and D) Thin-section electron micrographs of yeast endogenously expressing Mdm1 (C) or overexpressing Mdm1 (D). Bottom panels display traces of blue (the vacuole), red (nER), and yellow (NVJs). Bars, 500 nm. (E and F) Magnifications of the NVJs overexpressing Mdm1-GFP. (F) The mean distance between the nER membrane and the vacuole is 10.5 ± 1 nm. N, nucleus; V, vacuole. Bars, 100 nm.

Figure 4.

Mdm1 and Nvj3 localize to the NVJ in an Nvj1-independent manner. (A) Yeast expressing Nvj3-GFP and DsRed-HDEL (ER), with vacuoles (white). (B) Wild-type and mdm1Δ yeast expressing GFP-Nvj3. (C and D) nvj1Δ yeast expressing Mdm1-GFP and DsRed-HDEL (ER) and stained with CMAC. (E) Linear profile of Mdm1-GFP–positive NVJ in nvj1Δ yeast. (F and G) nvj1Δ yeast expressing GFP-Nvj3, DsRed-HDEL (ER), and vacuoles (white). (H) Linear profile of a GFP-Nvj3–positive NVJ in nvj1Δ yeast. The linear profiles in E and H are from single cells that are highly representative of many cells from at least three independent experiments. White dotted lines denote the cell periphery. AU, arbitrary units; N, nucleus; V, vacuole. Bars, 5 µm.

Figure 5.

SNX14 disease-analogous Mdm1 alleles perturb yeast sphingolipid metabolism. (A) Yeast with labeled vacuoles (FM4-64) under nitrogen starvation undergo PMN that consumes Mdm1-GFP. Arrows show PMN invaginations. (B) Yeast expressing a series of Mdm1-GFP truncations, DsRed-HDEL, and CMAC (white; vacuole). (C, left) Domain architecture of human Snx14 and its disease-associated alleles compared with Mdm1 truncations. (right) Correlative plating assay of yeast overexpressing Mdm1 truncations on media ± 500 ng/ml myriocin. The red box indicates RGS. (D) Model for the yeast NVJ showing novel proteins Nvj3 and Mdm1. Question marks denote hypothetical interactions. AR, armadillo repeats; N, nucleus; OBD, oxysterol binding domain; PH, pleckstrin homology; SMP, synaptotagmin-like mitochondrial lipid-binding protein domain; V, vacuole. Bars, 5 µm.