Biology subjects

Ou, C.

Publications and source records attributed to Ou, C..

3 recordsLinked to original sources

bioRxiv2023

Mechanism and cellular function of direct membrane binding by the ESCRT and ERES-associated Ca2+-sensor ALG-2

Apoptosis Linked Gene-2 (ALG-2) is a multifunctional intracellular Ca2+ sensor and the archetypal member of the penta-EF hand protein family. ALG-2 functions in the repair of damage to both the plasma and lysosome membranes and in COPII-dependent budding at endoplasmic reticulum exit sites (ERES). In the presence of Ca2+, ALG-2 binds to ESCRT-I and ALIX in membrane repair and to SEC31A at ERES. ALG-2 also binds directly to acidic membranes in the presence of Ca2+ by a combination of electrostatic and hydrophobic interactions. By combining GUV-based experiments and molecular dynamics simulations, we show that charge-reversed mutants of ALG-2 at these locations disrupt membrane recruitment. ALG-2 membrane binding mutants have reduced or abrogated ERES localization in response to Thapsigargin-induced Ca2+ release but still localize to lysosomes following lysosomal Ca2+ release. In vitro reconstitution shows that the ALG-2 membrane-binding defect can be rescued by binding to ESCRT-I. These data thus reveal the nature of direct Ca2+-dependent membrane binding and its interplay with Ca2+-dependent protein binding in the cellular functions of ALG-2.

biophysics↗

bioRxiv2023

Mutant Ppbbx24-del gene positively regulates light-induced anthocyanin accumulation in the red pear

Anthocyanins are pigments and nutrients in red pears regulated by BBX family genes. Herein, we characterized a 14-nucleotide deletion mutation in the coding region of the PpBBX24 gene from Red Zaosu pear (Pyrus pyrifolia White Pear Group), named Ppbbx24-del. Genetic and biochemical approaches were used to compare the roles of PpBBX24 and Ppbbx24-del in anthocyanin accumulation. Ppbbx24-del played a positive role in anthocyanin biosynthesis of the Red Zaosu pear peel by light treatment. Functional analyses based on overexpression in tobacco and transient overexpression in pear fruit peels showed that Ppbbx24-del promoted anthocyanin accumulation. Cyanidin and peonidin were major differentially expressed anthocyanins, and transcript levels of some structural genes in the anthocyanin biosynthesis pathway were significantly increased. Protein interaction assays showed that PpBBX24 was located in the nucleus and interacted with PpHY5, whereas Ppbbx24-del was colocalized in the nucleoplasm and did not interact with PpHY5. PpHY5 and Ppbbx24-del had positive regulatory effects on the expression of PpCHS, PpCHI, and PpMYB10 when acting alone, but had cumulative effects on gene activation when acting simultaneously. Alone, PpBBX24 had no significant effect on the expression of PpCHS, PpCHI, or PpMYB10, whereas it inhibited the activation effects of PpHY5 on downstream genes when it existed with PpHY5. Our study demonstrated that mutant Ppbbx24-del positively regulates the anthocyanin accumulation in pear. The results of this study clarify the mechanism and enrich the regulatory network of anthocyanin biosynthesis, which lays a theoretical foundation for Ppbbx24-del use to create red pear cultivars.

molecular biology↗

bioRxiv2022

In vitro reconstitution of calcium-dependent recruitment of the human ESCRT machinery in lysosomal membrane repair

The endosomal sorting complex required for transport (ESCRT) machinery has been shown to be centrally involved in repair of damage to both the plasma and lysosome membranes. ESCRT recruitment to sites of damage occurs on a fast time scale, and Ca2+ has been proposed to play a key signaling role in the process. Here, we show that the Ca2+-binding regulatory protein ALG-2 binds directly to negatively charged membranes in a Ca2+-dependent manner. Next, by monitoring the colocalization of ALIX with ALG-2 on negatively charged membranes, we show that ALG-2 recruits ALIX to the membrane. Furthermore, we show that ALIX recruitment to membrane orchestrates the downstream assembly of late-acting CHMP4B, CHMP3, CHMP2A subunits along with the AAA+ ATPase VPS4B. Finally, we show that ALG-2 can also recruit the ESCRT-III machinery to the membrane via the canonical ESCRT-I/II pathway. Our reconstitution experiments delineate the minimal sets of components needed to assemble the entire membrane repair machinery and open a new avenue for mechanistic understanding of endolysosomal membrane repair. Significance statementOne of the ways by which protein aggregates can propagate and lead to progression of a neurodegenerative disease is by damaging the membrane that is destined to degrade the misfolded, aggregated protein. ESCRT machinery has been implicated in sealing these damaged membranes, and the nature of the membrane recruitment trigger signal for this machinery is a major open question. Here, we show in vitro that ALG-2 can bring ESCRT machinery to membranes in a Ca2+-dependent manner.

cell biology↗