Biology subjects

Odorizzi, G.

Publications and source records attributed to Odorizzi, G..

7 recordsLinked to original sources

bioRxiv2026

AP-3 and the V-ATPase Modulate CTP Synthase Assembly Through Spatial Association at the Yeast Vacuole

The compartmentalization of metabolic enzymes into membraneless filaments termed cytoophidia represents a conserved regulatory mechanism, exemplified by cytidine triphosphate synthase (CTP synthase). CTP synthase assembles into pH-sensitive cytoophidia in the cytosol. In Saccharomyces cerevisiae, nutritional deprivation both triggers CTP synthase cytoophidia assembly and disassembles the vacuolar H{square}-ATPase (V-ATPase) that acidifies vacuoles (lysosomes), yet whether these processes are functionally linked remains unknown. We demonstrate spatial proximity between the yeast CTP synthase homolog Ura7, the V-ATPase, and the AP-3 adaptor complex that mediates vesicular transport to vacuoles. We show Ura7 localizes to vacuoles under both nutrient-rich and starvation conditions. Genetic disruption of AP-3 function altered Ura7 assembly dynamics under starvation, reducing total structures yet dramatically enhancing Ura7 cytoophidia elongation ([~]5-fold), revealing a dual regulatory role for AP-3 that both promotes Ura7 assembly and restrains elongation. Moreover, combining nutritional and pharmacological V-ATPase inhibition triggered massive Ura7 cytoophidia formation. These findings reveal a previously unrecognized spatial coupling between metabolic enzyme compartmentalization, vacuolar trafficking, and the pH regulation machinery, suggesting a new organizational principle whereby CTP synthase assembly dynamics respond to vacuolar function.

cell biology↗

bioRxiv2026

Septins associate with AP-3 to support trafficking to the vacuole/lysosome in yeast

Adaptor protein complex 3 (AP-3) mediates clathrin-independent transport to lysosomes, yet accessory factors supporting this pathway remain incompletely defined. In Saccharomyces cerevisiae, the C-terminal intrinsically disordered regions (IDRs) of both AP-3 large subunits ({delta} and {beta}3) serve as platforms for association with accessory factors. Through proteomic analysis of proteins associated with these IDRs, we identify the septin cytoskeleton as a candidate AP-3-associated factor. Bimolecular fluorescence complementation (BiFC) reveals a hierarchical pattern of association: AP-3 shows preferential proximity to core septin subunits (Cdc10, Cdc3, Cdc12) over terminal subunits (Cdc11 and Shs1). These terminal subunits serve as alternative caps of septin octamers, generating structurally distinct assemblies. Significantly, dysfunction of Cdc11 but not Shs1 selectively impairs AP-3-dependent cargo sorting without affecting the parallel vacuolar protein sorting (VPS) pathway to the vacuole (lysosome in yeast), providing genetic evidence for a specific functional connection between Cdc11-containing septin assemblies and AP-3-mediated transport.

cell biology↗

bioRxiv2025

Wbm0152, an outer membrane lipoprotein of the Wolbachia endosymbiont of Brugia malayi, inhibits yeast ESCRT complex activity

Human pathogenic filarial nematodes of the family Onchocercidae, including Brugia malayi and Onchocerca volvulus, cause debilitating filarial diseases such as lymphatic filariasis and river blindness. These mosquito-borne pathogens are obligately colonized by the gram-negative intracellular alphaproteobacterium, Wolbachia pipientis, which is essential for nematode sexual reproduction, long-term survival, and pathogenicity in the mammalian host. Like many intracellular bacteria, Wolbachia likely uses numerous surface-exposed and secreted effector proteins to regulate its ability to persist and replicate within nematode host cells. However, due to the inability to cultivate Wolbachia in the laboratory and the genetic intractability of both filarial nematodes and the bacterium, the molecular underpinnings that define the bacterium:nematode relationship are almost completely unknown. In this work, we show that the expression of a Wolbachia outer membrane lipoprotein, wBm0152, in Saccharomyces cerevisiae inhibits the activity of the conserved Endosomal Sorting Complex Required for Transport (ESCRT) complex and strongly disrupts endosomal maturation, leading to defects in ubiquitylated protein turnover. Using in vivo bimolecular fluorescence complementation, we find that Wbm0152 interacts with the Vps2p subunit of the ESCRT-III subcomplex as well as the Vps2p ortholog (BmVps2, Bm6583b) from a Wolbachia host nematode, Brugia malayi. These data suggest a novel role of ESCRT in Wolbachia persistence providing insight into the elusive relationship between these two organisms. AUTHOR SUMMARYFilarial diseases of mammals, including lymphatic filariasis and canine heartworm, are caused by vector-borne filarial nematodes of the family Onchocercidae. Many of the nematodes in this family are obligately colonized by an intracellular bacterium, Wolbachia pipientis, which is essential for the nematodes long-term survival, reproduction, and pathogenicity. Therefore, understanding the mechanisms used by Wolbachia to persist and replicate within host cells could provide new molecular targets for treating filarial infections. Due to the genetic intractability of both nematode and bacterium, however, significant progress on characterizing these interactions have proven difficult. In this work, we show that a predicted outer membrane lipoprotein, Wbm0152, of the Wolbachia endosymbiont of Brugia malayi inhibits yeast Endosomal Sorting Complex Required for Transport (ESCRT) complex activity in vivo. Wbm0152 interacts with a core subunit of the yeast ESCRT-III complex, as well as with the orthologous ESCRT-III protein from Brugia. ESCRTs are conserved across eukaryotes and are important for diverse cellular processes such as endosomal maturation, autophagy, and cellular division. As Wolbachia persists within a membrane-bound compartment within Brugia and must avoid host autophagic pathways, this study presents a potential mechanism by which Wolbachia may regulate Brugia membrane trafficking pathways to ensure its intracellular survival.

microbiology↗

bioRxiv2023

A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intralumenal vesicles (ILVs) at endosomes by Endosomal Sorting Complex Required for Transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB formation.

cell biology↗

bioRxiv2023

Mitochondrial-Derived Compartments are Multilamellar Domains that Encase Membrane Cargo and Cytosol

Preserving the health of the mitochondrial network is critical to cell viability and longevity. To do so, mitochondria employ several membrane remodeling mechanisms, including the formation of mitochondrial-derived vesicles (MDVs) and compartments (MDCs) to selectively remove portions of the organelle. In contrast to well-characterized MDVs, the distinguishing features of MDC formation and composition remain unclear. Here we used electron tomography to observe that MDCs form as large, multilamellar domains that generate concentric spherical compartments emerging from mitochondrial tubules at ER-mitochondria contact sites. Time-lapse fluorescence microscopy of MDC biogenesis revealed that mitochondrial membrane extensions repeatedly elongate, coalesce, and invaginate to form these compartments that encase multiple layers of membrane. As such, MDCs strongly sequester portions of the outer mitochondrial membrane, securing membrane cargo into a protected domain, while also enclosing cytosolic material within the MDC lumen. Collectively, our results provide a model for MDC formation and describe key features that distinguish MDCs from other previously identified mitochondrial structures and cargo-sorting domains. SUMMARYWilson and colleagues use electron tomography and time-lapse fluorescence microscopy to observe that mitochondrial-derived compartments (MDCs) are generated from outer mitochondrial membrane extensions that repeatedly elongate, coalesce, and invaginate to secure membrane cargo and cytosol within a distinct, protected domain.

cell biology↗

bioRxiv2020

Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT-driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologs in licensing ESCRT-III-dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1/Vps4/ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition. SummaryCargo sorting is coordinated with intralumenal vesicle budding during ESCRT-mediated multivesicular body biogenesis. Bro1 V domain stimulates Vps4 to promote ESCRT-III-driven intralumenal vesicle formation in a manner required for this coordinated process.

cell biology↗

bioRxiv2018

Plate-based assay of AP-3 traffic in budding yeast

SYNOPSISThe AP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. Here, a synthetic reporter is presented that allows convenient monitoring of AP-3 traffic, and direct screening or selection for mutants with defects in the pathway. The reporter can be assayed by fluorescence microscopy or in liquid or agar plate formats and is adaptable to high-throughput screening. SUMMARYAP-3 (adaptor complex 3) mediates traffic from the late Golgi or early endosomes to late endosomal compartments. In mammals, mutations in AP-3 cause Hermansky-Pudlak Syndrome type 2, cyclic neutropenias, and a form of epileptic encephalopathy. In budding yeast, AP-3 carries cargo directly from the trans-Golgi to the lysosomal vacuole. Despite the pathways importance and its discovery two decades ago, rapid screens and selections for AP-3 mutants have not been available. We now report GNSI, a synthetic, genetically encoded reporter that allows rapid plate-based assessment of AP-3 functional deficiency, using either chromogenic or growth phenotype readouts. This system identifies defects in both the formation and consumption of AP-3 carrier vesicles and is adaptable to high-throughput screening or selection in both plate array and liquid batch culture formats. Episomal and integrating plasmids encoding GNSI have been submitted to the Addgene repository.

cell biology↗