Biology subjects

Langemeyer, L.

Publications and source records attributed to Langemeyer, L..

3 recordsLinked to original sources

bioRxiv2023

Regulatory sites in the Mon1-Ccz1 complex control Rab5 to Rab7 transition and endosome maturation

Maturation from early to late endosomes depends on the exchange of their marker proteins Rab5 to Rab7. This requires Rab7 activation by its specific guanine nucleotide exchange factor (GEF) Mon1-Ccz1. Efficient GEF activity of this complex on membranes depends on Rab5, thus driving Rab-exchange on endosomes. However, molecular details on the role of Rab5 in Mon1-Ccz1 activation are unclear. Here we identify key features in Mon1 involved in GEF regulation. We show that the intrinsically disordered N-terminal domain of Mon1 autoinhibits Rab5-dependent GEF-activity on membranes. Consequently, Mon1 truncations result in higher GEF activity in vitro, and a shift from Rab5 to more Rab7 positive structures in Drosophila nephrocytes and yeast, suggesting faster endosomal maturation. Using modeling, we further identify a conserved Rab5 binding site in Mon1. Mutations impairing Rab5 interaction result in poor GEF activity on membranes and growth defects in vivo. Our analysis provides a framework to understand the mechanism of Rab-conversion and organelle maturation along the endomembrane system. SummaryTransport of proteins via the endolysosomal pathway requires Rab5 on early endosomes, which is replaced by Rab7 on late endosomes. Here, we identify distinct regulatory sites in the Rab7 activator, the Mon1-Ccz1 complex, shedding light on the regulation of Rab5 to Rab7 transition.

cell biology↗

bioRxiv2022

Targeting of the Mon1-Ccz1 Rab guanine nucleotide exchange factor to distinct organelles by a synergistic protein and lipid code

Activation of the small GTPase Rab7 by its cognate guanine nucleotide exchange factor (GEF) Mon1-Ccz1 (MC1) is a key step in the maturation of endosomes and autophagosomes. This process is tightly regulated and subject to precise spatiotemporal control of MC1 localization. We here identify and characterize an amphipathic helix in Ccz1, which is required for the function of Mon-Ccz1 in autophagy, but not endosomal maturation. Furthermore, our data show that the interaction of the Ccz1 amphipathic helix with lipid packing defects, binding of Mon1 basic patches to positively charged lipids and association of MC1 with recruiter proteins collectively govern membrane recruitment of the complex in a synergistic and redundant manner. The data demonstrate that specific protein and lipid cues convey the differential targeting of MC1 to endosomes and autophagosomes. We reveal the molecular mechanism how MC1 is adapted to recognizes distinct target compartments by exploiting the unique biophysical properties of organelle membranes and thus provide a model how the complex is regulated and activated independently in different functional contexts.

biochemistry↗

bioRxiv2022

Structure of the lysosomal membrane fusion machinery

Lysosomes are of central importance in cellular recycling, nutrient signaling 1,2 and endocytosis, and are tightly connected to autophagy 3 and the invasion of pathogenic bacteria and viruses 1,4. Lysosomal fusion events are fundamental to cell survival and require HOPS, a conserved heterohexameric tethering complex 5,6. HOPS recognizes and binds small membrane-associated GTPases on lysosomes and organelles, and assembles membrane bound SNAREs for fusion 7,8. Through tethering, HOPS brings membranes in close proximity to each other and significantly increases fusion efficacy by catalysing SNARE assembly. Consequently, different HOPS mutations are causative for severe diseases 6. Despite its fundamental cellular duties, it remained speculative how HOPS fulfils its function as high-resolution structural data were unavailable. Here, we used cryo-electron microscopy to reveal the structure of HOPS. In the complex, two central subunits form the backbone and an assembly hub for the functional domains. Two GTPase binding units extend to opposing ends, while the SNARE binding module points to the side, resulting in a triangular shape of the complex. Unlike previously reported, HOPS is surprisingly rigid and extensive flexibility is confined to its extremities. We show that HOPS complex variants with mutations proximal to the backbone can still tether membranes but fail to efficiently promote fusion indicating, that the observed integrity of HOPS is essential to its function. In our model, the core of HOPS acts as a counter bearing between the flexible GTPase binding domains. This positions the SNARE binding module exactly between the GTPase anchored membranes to promote fusion. Our structural and functional analysis reveals the link between the spectacular architecture of HOPS and its mechanism that couples membrane tethering and SNARE assembly, to catalyse lysosomal fusion.

biophysics↗