Ivanovic, L. | bioRxiv Science

bioRxiv2025

The molecular basis of tricalbin-mediated membrane contact site organization in cells

Membrane contact sites facilitate molecular exchanges through physical interactions between organelles, connected by specific protein tethers. Among these tethers are the tricalbins, which mediate contacts between endoplasmic reticulum (ER) and plasma membrane in yeast. Tricalbins are integral to the ER, have a cytosolic lipid binding domain and bind the plasma membrane through C2 domains. Here, we combine fluorescence recovery after photobleaching with correlative light and 3D electron microscopy to dissect how tricalbins control their localization, dynamic distribution and contact site organization. We find that heteromerization via lipid binding domains is a prerequisite for tricalbin accumulation at contact sites, membrane curvature sensing and restrained mobility in the ER. By altering tricalbin protein domains, we show that intermembrane distances and intrinsically disordered regions interdependently control distribution and dynamics of contact site tethers. Our study reveals principles of contact site architecture that are fine-tuned by tricalbin domain organization.

cell biology↗

bioRxiv2023

The punctate localisation of the yeast sterol transporter Ysp2p is determined by three dimerisation interfaces in its C-terminus

Sterol lipids traffic between intracellular compartments by vesicular and non-vesicular routes. Sterol traffic from the plasma membrane to the endoplasmic reticulum (ER), so-called retrograde traffic, particularly depends on a non-vesicular mechanism, being transported by the ubiquitous family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs, also called GRAMD1/Asters in humans, VASt in plants). LAMs are similar to many lipid transfer proteins in that they localise to membrane contact sites and carry lipids between two organelles. In yeast, the major LAM active at ER-plasma membrane contact sites is Ysp2p, which has a uniquely punctate distribution in the cortical ER. Here, we have comprehensively dissected how Ysp2p achieves its distinctive punctate localisation. We show that the PHGRAM domain of Ysp2p has membrane binding properties similar to its human counterpart GRAMD1B, but that this is not important for punctate localisation of Ysp2p. Instead, all regions necessary for the punctate localisation of Ysp2p at membrane contacts are present in [~]200 residues at the C-terminus of Ysp2p, with a critical region being a small y-sheet that we predict homodimerises. We also study the role of punctate localisation of Ysp2 in its function in retrograde sterol traffic, and show that function does not require the punctate localisation, but instead requires a polybasic region adjacent to the sterol transfer domain. Finally, to investigate the interaction of the polybasic region with the plasma membrane, we examine contacts populated by the Ysp2 C-terminus by electron tomography, and find that they consist of generic cortical ER.

cell biology↗