Biology subjects

Port, S. A.

Publications and source records attributed to Port, S. A..

3 recordsLinked to original sources

bioRxiv2020

The Structural Basis of Rubisco Phase Separation in the Pyrenoid

Approximately one-third of global CO2 fixation occurs in a phase separated algal organelle called the pyrenoid. Existing data suggest that the pyrenoid forms by the phase-separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase-separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein EPYC1 (Essential Pyrenoid Component 1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly-spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of one of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation, and pyrenoid formation. Cryo-electron tomography supports a model where EPYC1 and Rubisco form a co-dependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

molecular biology↗

bioRxiv2018

Munc18-1 catalyzes neuronal SNARE assembly by templating SNARE association

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. Next, SNAP-25 binds the templated SNAREs to form a partially-zippered SNARE complex. Finally, full zippering displaces Munc18-1. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

biophysics↗

bioRxiv2018

Mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, are of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are only rescued by expressing wild-type nup88 and not the disease-linked mutant forms of nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

cell biology↗