Figure 6.
Cnm1-mediated contact sites require Tom70. (A) Cnm1 is an outer nuclear membrane protein. A strain overexpressing Cnm1-GFP (OE Cnm1-GFP) during stationary phase shows areas where Cnm1 is not localized to mitochondria (stained by MitoTracker Orange) but does colocalize with the outer nuclear membrane (nuclear ER) visualized using a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL). Arrows mark areas where Cnm1-GFP signals colocalize with the nuclear ER signal, but not with the mitochondrial signal. Scale bar, 1 µm. (B) Loss of tom70 results in Cnm1 redistributing uniformly around the nucleus. Shown are strains overexpressing (OE) Cnm1-GFP on the background of Δtom70 or control cells, imaged in mid-logarithmic phase using MitoTracker Orange for mitochondrial staining. Scale bar, 5 µm. (C) Tom70 physically interacts with Cnm1. Pull-down of overexpressed Cnm1 tagged with GFP on its C terminus in strains expressing either Tom70 or Tom20 tagged with mCherry on their C termini. Coimmunoprecipitation (co-IP) samples were analyzed by Western blotting and probed with antibodies against GFP and mCherry. Input (10% of total immunoprecipitates) is shown. The number above each immunoprecipitation band represents the enrichment of the protein. (D) Overexpression (OE) of Cnm1 results in the accumulation of soluble GFP-Tom70 around the nuclear membrane. Overexpressed Tom70 whose TMD (1–38 aa) has been truncated and is tagged with GFP on its N terminus (OE GFP-Δtmd-Tom70) shows cytosolic distribution in control cells. Overexpression of Cnm1 concentrates the soluble Tom70 around the nuclear membrane marked by a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL). Mitochondria were dyed with MitoTracker Orange. Control and overexpressed Cnm1 strains are adjusted to different intensities. Scale bar, 5 µm. (E) Overexpressed (OE) GFP-Δtmd-Tom70 is fully colocalized with overexpressed Cnm1-mCherry on the nuclear periphery. Scale bar, 5 µm. (F) Deletion of the predicted iMTS-L sequence of Cnm1 (350–404 aa) abrogates mitochondrial clustering around the nucleus and results in redistribution of Cnm1 over the entire nuclear membrane. Cnm1-GFP (full length or mutant) were expressed under a TEF2 promoter. Mitochondria are dyed with MitoTracker Orange. Scale bar, 5 µm. (G) Soluble Cnm1 decorates the mitochondrial OM. Overexpressed Cnm1 truncated at its N terminus by fusion of a GFP molecule to remove its predicted TMD (1–112 aa; OE GFP-Δtmd-Cnm1) was expressed in either WT Tom70 cells or cells overexpressing Tom70 (OE Tom70) under the NOP1 promoter. The nuclear envelope is visualized by a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL; SS-BFP-HDEL in WT Tom70 and OE Tom70 strains is adjusted to different intensities). Mitochondria are marked by MitoTracker Orange. In control cells, GFP-Δtmd-Cnm1 shows cytosolic distribution as well as enrichment around the mitochondrial periphery and no nuclear periphery staining. Overexpression of Tom70 causes an even brighter signal to accumulate around mitochondria, suggesting that its levels are restrictive to Cnm1 recruitment to mitochondrial surfaces. Scale bar, 5 µm. (H) Schematic working model on Cnm1 activity in mediating nucleus–mitochondria contacts. PC levels regulate Cnm1 abundance in the cell. Cnm1 on the nuclear ER membrane interacts with Tom70 on the mitochondrial membrane.

Cnm1-mediated contact sites require Tom70. (A) Cnm1 is an outer nuclear membrane protein. A strain overexpressing Cnm1-GFP (OE Cnm1-GFP) during stationary phase shows areas where Cnm1 is not localized to mitochondria (stained by MitoTracker Orange) but does colocalize with the outer nuclear membrane (nuclear ER) visualized using a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL). Arrows mark areas where Cnm1-GFP signals colocalize with the nuclear ER signal, but not with the mitochondrial signal. Scale bar, 1 µm. (B) Loss of tom70 results in Cnm1 redistributing uniformly around the nucleus. Shown are strains overexpressing (OE) Cnm1-GFP on the background of Δtom70 or control cells, imaged in mid-logarithmic phase using MitoTracker Orange for mitochondrial staining. Scale bar, 5 µm. (C) Tom70 physically interacts with Cnm1. Pull-down of overexpressed Cnm1 tagged with GFP on its C terminus in strains expressing either Tom70 or Tom20 tagged with mCherry on their C termini. Coimmunoprecipitation (co-IP) samples were analyzed by Western blotting and probed with antibodies against GFP and mCherry. Input (10% of total immunoprecipitates) is shown. The number above each immunoprecipitation band represents the enrichment of the protein. (D) Overexpression (OE) of Cnm1 results in the accumulation of soluble GFP-Tom70 around the nuclear membrane. Overexpressed Tom70 whose TMD (1–38 aa) has been truncated and is tagged with GFP on its N terminus (OE GFP-Δtmd-Tom70) shows cytosolic distribution in control cells. Overexpression of Cnm1 concentrates the soluble Tom70 around the nuclear membrane marked by a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL). Mitochondria were dyed with MitoTracker Orange. Control and overexpressed Cnm1 strains are adjusted to different intensities. Scale bar, 5 µm. (E) Overexpressed (OE) GFP-Δtmd-Tom70 is fully colocalized with overexpressed Cnm1-mCherry on the nuclear periphery. Scale bar, 5 µm. (F) Deletion of the predicted iMTS-L sequence of Cnm1 (350–404 aa) abrogates mitochondrial clustering around the nucleus and results in redistribution of Cnm1 over the entire nuclear membrane. Cnm1-GFP (full length or mutant) were expressed under a TEF2 promoter. Mitochondria are dyed with MitoTracker Orange. Scale bar, 5 µm. (G) Soluble Cnm1 decorates the mitochondrial OM. Overexpressed Cnm1 truncated at its N terminus by fusion of a GFP molecule to remove its predicted TMD (1–112 aa; OE GFP-Δtmd-Cnm1) was expressed in either WT Tom70 cells or cells overexpressing Tom70 (OE Tom70) under the NOP1 promoter. The nuclear envelope is visualized by a BFP with a signal sequence and an ER retention signal (SS-BFP-HDEL; SS-BFP-HDEL in WT Tom70 and OE Tom70 strains is adjusted to different intensities). Mitochondria are marked by MitoTracker Orange. In control cells, GFP-Δtmd-Cnm1 shows cytosolic distribution as well as enrichment around the mitochondrial periphery and no nuclear periphery staining. Overexpression of Tom70 causes an even brighter signal to accumulate around mitochondria, suggesting that its levels are restrictive to Cnm1 recruitment to mitochondrial surfaces. Scale bar, 5 µm. (H) Schematic working model on Cnm1 activity in mediating nucleus–mitochondria contacts. PC levels regulate Cnm1 abundance in the cell. Cnm1 on the nuclear ER membrane interacts with Tom70 on the mitochondrial membrane.

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