Biology subjects

Merz, A. J.

Publications and source records attributed to Merz, A. J..

10 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

β-strand complementation within tip initiation complexes licenses assembly of diverse type IV filaments

PilC/PilY1 proteins are tip-located adhesins of type IV pili (T4P) that are critical for T4P function in diverse behaviors including twitching motility, DNA uptake, and host cell adhesion. PilC and PilY1 adhesins are proposed to interact with initiation complexes composed of minor pilins (PilIJK family proteins) to aid in initiation of T4P polymerization, but it has been unclear how PilC/PilY1 proteins promote fiber assembly. We combined structural modeling, genetic, and biochemical experiments using Neisseria gonorrhoeae and Acinetobacter baylyi to delineate how PilC/PilY1 control T4P assembly: a short peptide at the C-terminus of PilC/PilY1 initiates T4P assembly via {beta}-strand complementation with PilK-family minor pilins. This {beta}-strand is necessary and partially sufficient to trigger fiber assembly. In a working model, the PilK-PilC/PilY1 complex is recognized by a preformed PilI-PilJ heterodimer to form a quaternary "licensing complex" that then templates and initiates fiber assembly. In type II secretion systems (T2SS) lacking PilC/PilY1, PilK homologs directly incorporate the terminal {beta}-strand provided by PilC/PilY1 in T4P. Moreover, phylogenetically distinct Tad T4P lack a canonical PilK homolog and instead contain a structurally similar minor pilin-like protein called TadG/CpaL that is important for fiber assembly. We show that CpaL of Caulobacter crescentus Tad T4P acts similarly to the T2SS PilK homolog to provide the C-terminal {beta}-strand required for assembly. Our results explain how PilC/PilY1 can be retained on the fiber tip under enormous tensile loads generated during mechanical shear and T4P retraction and demonstrate how diverse T4P systems employ {beta}-strand complementation to license fiber assembly. SIGNIFICANCEProkaryotic type IV filaments are ancient, diverse, and broadly distributed nanomachines that assemble and retract to execute diverse microbial functions. They include type IV pili and type II secretion systems, mediating toxin secretion, motility, surface adhesion, biofilm formation, DNA uptake, and other functions. Here, we show that two widely conserved subunits of the tip, PilI and PilJ, form a module that recognizes the folding of a {beta}-sheet in a third subunit, PilK. The final {beta}-strand in this sheet can be supplied in trans by the last [~]10 aminoacyl residues of large PilC/PilY1 adhesins, or in cis by PilK itself. In a working model, this recognition results in formation of a PilIJK trimer, which then licenses fiber polymerization through a templating mechanism.

microbiology↗

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↗

bioRxiv2026

Sequential restriction of SNARE-mediated fusion by the COPII inner coat and SNARE chaperones

SNARE-mediated fusion requires assembly of four SNARE domains (R, Qa, Qb, and Qc) distributed across two membranes, with an SM (Sec1/Munc-18 family) chaperone catalyzing this assembly. We investigated topological requirements for the four SNAREs that mediate ER-Golgi fusion using in vitro assays. In the presence of the cognate SM Sly1, we find that a single topology drives efficient fusion: the R SNARE on one membrane versus Qa/Qb/Qc SNAREs on the other. These results prompted us to look upstream, at COPII-SNARE interactions. The assembled COPII coat is known to block fusion. We discovered that of five COPII core subunits, the Sar1 GTPase and Sec23/Sec24 subunits were necessary and sufficient to prevent fusion for prolonged periods, and that Sar1 was dispensable for inhibition over short periods. When Sec24- interactions with vesicle SNAREs were disrupted, inhibition was relieved, indicating that SNARE sequestration by COPII prevents fusion. These observations help to explain how appropriate fusion events are facilitated while inappropriate events are deterred.

cell biology↗

bioRxiv2022

Remodeling of yeast vacuole membrane lipidomes from the log (1-phase) to stationary stage (2-phases)

Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micron-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids ([~]50%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These results fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols. SIGNIFICANCE STATEMENTWhen budding yeast shift from growth to quiescence, the membrane of one of their organelles (the vacuole) undergoes liquid-liquid phase separation. What changes in the membranes lipids cause this phase transition? Here, we conduct lipidomics of immuno-isolated vacuole membranes. We analyze our data in the context of lipid melting temperatures, inspired by observations that liquid-liquid phase separation in model membranes requires a mixture of lipids with high melting temperatures, lipids with low melting temperatures, and sterols. We find that phase-separated vacuole membranes have higher concentrations of PC lipids, and that those lipids have higher melting temperatures. To conduct our experiments, we developed a tagged version of a protein (Mam3) for immuno-isolation of vacuole membranes.

biophysics↗

bioRxiv2021

Yeast cells actively tune their membranes to phase separate at temperatures that scale with growth temperatures

Membranes of vacuoles, the lysosomal organelles in yeast, undergo extraordinary changes during the cells normal growth cycle. The cycle begins with a stage of rapid cell growth. Then, as glucose becomes scarce, growth slows, and the vacuole membranes phase-separate into micron-scale liquid domains. Recent studies suggest that these domains are important for yeast survival by laterally organizing membrane proteins that play a key role in a central signaling pathway conserved among eukaryotes (TORC1). An outstanding question in the field has been whether yeast stringently regulate the phase transition and how they respond to new physical conditions. Here, we measure transition temperatures - an increase of roughly 15{degrees}C returns vacuole membranes to a state that appears uniform across a range of growth temperatures. We find that broad populations of yeast grown at a single temperature regulate the transition to occur over a surprisingly narrow temperature range. Moreover, the transition temperature scales linearly with the growth temperature, demonstrating that the cells physiologically adapt to maintain proximity to the transition. Next, we ask how yeast adjust their membranes to achieve phase separation. Specifically, we test how levels of ergosterol, the main sterol in yeast, induce or eliminate membrane domains. We isolate vacuoles from yeast during their rapid stage of growth, when their membranes do not natively exhibit domains. We find that membrane domains materialize when ergosterol is depleted, contradicting the assumption that increases in ergosterol cause membrane phase separation in vivo, and in agreement with prior studies that use artificial and cell-derived membranes. SIGNIFICANCE STATEMENTPhase separation in membranes creates domains enriched in specific components. To date, the best example of micron-scale phase separation in the membrane of an unperturbed, living cell occurs in a yeast organelle called the vacuole. Recent studies indicate that the phases are functionally important, enabling yeast survival during periods of cellular stress. We have discovered that yeast regulate this phase transition; the temperature at which membrane components mix into a single phase is ~15{degrees}C above the growth temperature. To maintain this offset, yeast may tune the level of ergosterol (a molecule that is structurally similar to cholesterol) in their membranes. We find that reducing sterol levels in vacuole membranes causes them to phase separate, in contrast to previous assumptions.

biophysics↗

bioRxiv2020

Gatekeeper helix activates Golgi SM protein Sly1 and directly mediates close-range vesicle tethering

The essential Golgi protein Sly1 is a member of the SM (Sec1/mammalian Unc-18) family of SNARE chaperones. Sly1 was originally identified through gain-of-function alleles that bypass requirements for diverse vesicle tethering factors. Employing genetic analyses and chemically defined reconstitutions of ER-Golgi fusion, we discovered that a loop conserved among Sly1 family members is not only autoinhibitory, but also acts as a positive effector. An amphipathic helix within the loop directly binds high-curvature membranes; membrane binding is required for relief of Sly1 autoinhibition and allows Sly1 to directly tether incoming vesicles to the Qa-SNARE on the target organelle. The SLY1-20 allele bypasses requirements for diverse tethering factors but loses this functionality if Sly1 membrane binding is impaired. We propose that long-range tethers, including Golgins and multisubunit tethering complexes, hand off vesicles to Sly1, which then tethers at close range to activate SNARE assembly and fusion in the early secretory pathway.

cell biology↗

bioRxiv2020

Golgi SM protein Sly1 promotes productive trans-SNARE complex assembly through multiple mechanisms

SNARE chaperones of the Sec1/mammalian Unc-18 (SM) family have critical roles in SNARE-mediated membrane fusion. Using SNARE and Sly1 mutants, and a new in vitro assay of fusion, we separate and assess proposed mechanisms through which Sly1 augments fusion: (i) opening the closed conformation of the Qa-SNARE Sed5; (ii) close-range tethering of vesicles to target organelles, mediated by the Sly1-specific regulatory loop; and (iii) preferential nucleation of productive trans-SNARE complexes. We show that all three mechanisms are important and operate in parallel, and we present evidence that close-range tethering is particularly important for trans-complex assembly when cis-SNARE assembly is a competing process. In addition, the autoinhibitory N-terminal Habc domain of Sed5 has at least two positive activities: the Habc domain is needed for correct Sed5 localization, and it directly promotes Sly1-dependent fusion. Remarkably, "split Sed5," with the Habc domain present only as a soluble fragment, is functional both in vitro and in vivo.

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↗

bioRxiv2017

The Dense-Core Vesicle Maturation Protein CCCP-1 Binds both RAB-2 and Membranes through a Conserved C-terminal Coiled-coil Domain

Dense-core vesicles (DCVs) are secretory organelles that store and release modulatory neurotransmitters from neurons and endocrine cells. Recently, the conserved coiled-coil protein CCCP-1 was identified as a component of the DCV biogenesis pathway in the nematode C. elegans. CCCP-1 binds the small GTPase RAB-2 and colocalizes with it at the trans-Golgi. Here we report a structure-function analysis of CCCP-1 to identify domains of the protein important for its localization, binding to RAB-2, and function in DCV biogenesis. We find that the CCCP-1 C-terminal domain (CC3) has multiple activities. CC3 is necessary and sufficient for CCCP-1 localization and for binding to RAB-2, and is required for the function of CCCP-1 in DCV biogenesis. Additionally, CCCP-1 binds membranes directly through its CC3 domain, indicating that CC3 may comprise a previously uncharacterized lipid-binding motif. We conclude that CCCP-1 is a coiled-coil protein that binds an activated Rab and localizes to the Golgi via its C-terminus, properties similar to members of the golgin family of proteins. CCCP-1 also shares biophysical features with golgins; it has an elongated shape and forms oligomers.\n\nSynopsis statementCCCP-1 is a coiled-coil protein important for dense-core vesicle (DCV) biogenesis. A structure-function analysis of CCCP-1 shows that its C-terminal domain is required for (1) localization to membrane compartments near the trans-Golgi, (2) binding to activated RAB-2, (3) function in DCV biogenesis, and (4) direct binding to membranes. CCCP-1 has an elongated shape and forms oligomers. These findings suggest that CCCP-1 resembles members of the golgin family of proteins that act as membrane tethers.

biochemistry↗