Lam6 Regulates the Extent of Contacts between Organelles

Abstract

Communication between organelles is crucial for eukaryotic cells to function as one coherent unit. An important means of communication is through membrane contact sites, where two organelles come into close proximity allowing the transport of lipids and small solutes between them. Contact sites are dynamic in size and can change in response to environmental or cellular stimuli; however, how this is regulated has been unclear. Here, we show that Saccharomyces cerevisiae Lam6 resides in several central contact sites: ERMES (ER/mitochondria encounter structure), vCLAMP (vacuole and mitochondria patch), and NVJ (nuclear vacuolar junction). We show that Lam6 is sufficient for expansion of contact sites under physiological conditions and necessary for coordination of contact site size. Given that Lam6 is part of a large protein family and is conserved in vertebrates, our work opens avenues for investigating the underlying principles of organelle communication.

Graphical abstract

Figure 1

Lam6 Is an Uncharacterized Binding Partner of the ERMES Complex (A–D) Pull-down of HA-tagged ERMES components (Mdm10, A; Mdm12, B; Mdm34, C; and Mmm1, D) uncovered that all ERMES components interact with the same uncharacterized protein, Lam6. Lam6 and members of the ERMES complex are marked in green. (E–F) Reciprocal pull-down demonstrated that endogenously expressed C-terminally tagged Lam6 (E) and overexpressed N-terminally tagged Lam6 (F) have the same strong binding partners that include the ERMES complex members (G). GFP-tagged ERMES complex proteins retain their characteristic punctate structure in Δlam6, suggesting that Lam6 is not an essential complex member. Scale bar represents 5 μm.

Figure 2

Lam6 Is Localized to Several Cellular Contact Sites (A) Fluorescence microscopy demonstrates that Lam6-GFP co-localizes with ERMES (Mdm34-Cherry) (yellow arrows) and also localizes to non-ERMES locations in the cell (red arrows). These additional locations co-localized with the NVJ (Nvj1-GFP) as well as with the vCLAMP (GFP-Vps39). Scale bar represents 5 μm. (B) Overexpression and tagging of Lam6 confirmed that it co-localizes with ERMES (Mdm34-GFP) (yellow arrows) as well as to additional contact sites (red arrows), NVJ (marked by Nvj1) and vCLAMP (marked by Vps39). Scale bar represents 5 μm.

Figure 3

Overexpression and Tagging of Lam6 Affects the Extent of Contact Sites (A) Fluorescence microscopy demonstrates that the overexpression of Cherry-Lam6 (OE-Cherry-LAM6) results in an expansion of the following three contact sites: ERMES (Mdm34-GFP), NVJ (Nvj1-GFP), and vCLAMP (GFP-Vps39). This suggests that an increase in Lam6 levels in the contact site is sufficient for its expansion. The numbers represent the average contact site size (120 cells per sample). Scale bar represents 5 μm. (B) Immuno-EM verified that GFP-Lam6 overexpression indeed causes an expansion of contact site size. While vCLAMP was hardly visible in WT cells, it could be detected easily in the OE strain (albeit often had ER tubules invading it). The NVJ underwent expansion as well as evoked PMN in OE strains, and the ER-mitochondria contact became large and elongated instead of small and distinct. N, nucleus; M, mitochondria; V, vacuole. Scale bar represents 200 nm (see also Figure S3I).

Figure 4

Lam6 Is Important for Cross-Talk between Contact Sites (A) Fluorescent microscopy shows that the ERMES contact (as measured by the number of Mdm34-GFP puncta per cell) expanded in the Δvps39 background relative to WT. However, the expansion did not occur on the background of Δvps39 Δlam6, demonstrating that Lam6 is necessary for ERMES expansion under these conditions. Scale bar represents 5 μm. (B) Quantitation of (A). Bars represent the percentage of cells containing the specific number of puncta/cell out of total cells counted for the strain (for WT, n = 234 cells; for Δvps39, n = 246; for Δvps39 Δlam6, n = 271). (C) Fluorescent microscopy demonstrates that downregulating ERMES contacts (by growing GALp-MDM34 strains in glucose) indeed caused expansion of vCLAMP (GFP-Vps39). However, this expansion was diminished in a Δlam6 background. Scale bar represents 5 μm (see also Figure S4). (D) A model summarizing our hypothesis on the way that Lam6 functions to regulate contact site communication. In normal cells, where all contact sites are intact, Lam6 is found mostly in ERMES contact sites, and to a lesser extent in vCLAMP and NVJ. However, in case a contact site is lost, the Lam6 proteins that were localized to this contact become free to associate with the other contact sites, thus signaling that they must expand. Raising Lam6 levels in any other way in a contact site therefore would cause an expansion of this contact site.