Biology subjects

Ivanovic, L.

Publications and source records attributed to Ivanovic, L..

3 recordsLinked to original sources

bioRxiv2026

Adaptor interactions trigger Pan1 self-assembly during clathrin-mediated endocytosis in budding yeast

Clathrin-mediated endocytosis requires the coordinated assembly of a highly dynamic protein network that couples membrane remodeling to actin-driven force generation. The essential budding yeast protein, Pan1, scaffolds the endocytic protein network by linking adaptor proteins, and other coat components, and actin assembly regulators. How Pan1 coordinates all these different proteins in space and time is not well understood. We show that Pan1 undergoes biomolecular condensation. Elevated Pan1 expression induced the formation of condensates that exhibited partial rapid molecular exchange, temperature-dependent reversibility, and sensitivity to disruption of weak hydrophobic interactions. The Pan1 assemblies are compositionally selective, preferentially enriching late-stage endocytic factors while excluding early adaptor proteins. Truncation analysis demonstrated that condensation depends on cooperative contributions from intrinsically disordered regions, the EH2 domain, and the oligomerization module, whereas a C-terminal region negatively regulates condensation. Deletion of Pan1 regions promoting its self-assembly perturbed Pan1 assembly and function at endocytic sites. Disruption of interactions between Pan1 and endocytic adaptors did not alter the dynamics of endocytic events but instead reduced the number of endocytic events marked by Pan1 and caused the formation of ectopic Pan1 condensates. This indicates that adaptor-mediated interactions spatially constrain Pan1 localization by seeding Pan1 assembly at endocytic sites. Our findings support a model in which adaptor proteins seed Pan1 assembly at the endocytic sites while multivalent interactions drive Pan1 self-assembly to build the higher-order molecular network of the late endocytic coat.

cell biology↗

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↗