Molecular Biology of the Cell

At the core of cilia are microtubules which are important for establishing length and assisting ciliary assembly and disassembly; however, another role for microtubule regulation on ciliogenesis lies outside of the cilium. The microtubule cytoskeleton is a highly dynamic structure which polymerizes and depolymerizes rapidly to assist in cellular processes. These processes have been studied across various organisms with chemical as well as genetic perturbations. However, these have generated conflicting data in terms of the role of cytoplasmic microtubules (CytoMTs) and free tubulin dynamics during ciliogenesis. Here we look at the relationship between ciliogenesis and cytoplasmic microtubule dynamics in Chlamydomonas reinhardtii using chemical and mechanical perturbations. We find that not only can stabilized CytoMTs allow for normal ciliary assembly, but high calcium concentrations and low pH-induced deciliation cause CytoMTs to depolymerize separately from ciliary shedding. In addition, we find that ciliary shedding through mechanical shearing, cilia regenerate earlier despite intact CytoMTs. Our data suggests that cytoplasmic microtubules are not a sink for a limiting pool of cytoplasmic tubulin in Chlamydomonas, depolymerization that occurs following deciliation is a consequence rather than a requirement for ciliogenesis, and intact CytoMTs in the cytoplasm and the proximal cilium support more efficient ciliary assembly.

Cells maintain an appropriate size to function, yet the mechanisms that enable size adaptation to environmental stress remain poorly understood. Fission yeast cells enter mitosis and divide at a threshold size when cyclin-dependent kinase (Cdk1) is activated through size- and time-dependent scaling of its regulators: Cdr2 kinase with cell surface area, Cdc25 phosphatase with cell volume, and mitotic cyclin Cdc13 with cell cycle time. This integrated size control network is characterized in nutrient-rich conditions, but under stress it remains unclear which size parameters cells monitor, and which size- or time-sensing pathways mediate adaptation. Using high-throughput image analysis, we quantified the geometry of dividing cells under osmotic, oxidative, and low glucose conditions. Wild-type cells increased their surface area-to-volume (SA:Vol) ratio in low glucose but decreased it under osmotic or oxidative stress, revealing distinct geometric strategies for environmental size adaptation. Genetic perturbations of size- and time-sensing pathways revealed that Cdc25 is required for volume-based adaptation to oxidative and osmotic stress, Cdc13 contributes to osmotic stress response, and Cdr2 promotes surface area-based expansion in low glucose. Although disrupting individual pathways altered normal geometric responses, cells remained viable, suggesting that a modular size control system enables flexible geometric adaptation to changing environments.

The conserved GTPase Cdc42 is a major regulator of polarized growth in most eukaryotes. In Schizosaccharomyces pombe, Cdc42 activity displays anticorrelated oscillatory dynamics between the growing ends enabling bipolarity. Cdc42 at each end is activated only when the opposite end loses activity. This suggests that a regulator of Cdc42 likely travels end-to-end to activate Cdc42. The oscillatory dynamics between the growing ends have also been observed in Cdc42 activator Scd1, its scaffold Scd2. It is unclear how these proteins move between the ends to facilitate bipolarity. We find that Scd1 does not travel between the cell ends via actin-mediated delivery. Instead, we show that Scd1 is mostly cytoplasmic and diffuses between the cell ends. The rate of diffusion is not entirely proportional to increasing the mass of Scd1 and cells lacking the inhibitor Pak1 kinase show decreased diffusion. Moreover, we show that Scd1 diffuses at a much faster rate compared to its scaffold Scd2. These findings suggest that Scd1 diffusion is not random and is regulated by Pak1 kinase. We find that decreasing the rate of diffusion disrupts Cdc42 oscillatory dynamics and results in monopolarity. Our results show that end-to-end Scd1 diffusion drives Cdc42 oscillatory dynamics and regulates cell polarity. SIGNIFICANCE STATEMENTO_LICdc42 activation shows oscillatory dynamics between the sites of growth C_LIO_LIThe Cdc42 GEF Scd1 diffuses from site of activation to the opposite end to facilitate these oscillatory dynamics C_LIO_LIThis diffusion is not random and likely depends on intrinsic properties of the Scd1 protein. C_LI

Understanding of the mechanisms that initiate clathrin-mediated endocytosis (CME) is incomplete. Recent studies in budding yeast identified the endocytic adaptor protein Yap1801/Yap1802 (budding yeast AP180) as a key CME factor that promotes CME initiation in daughter cells during polarized growth, but how Yap1801/2 is recruited preferentially to the plasma membrane of daughter cells is not clear. The only known cargos for Yap1801/2 in yeast are the synaptobrevins Snc1 and Snc2, which act as v-SNARES for exocytic vesicles arriving at the plasma membrane and are essential for polarized cell growth. In this study, we analyze the spatiotemporal dynamics of functional, fluorescently-tagged Snc1/2 expressed from their endogenous loci and provide evidence that, in concert with anionic phospholipids, Snc1/2 recruit Yap1801/2 preferentially to growing daughter cells. These findings suggest that the coincidence of anionic phospholipids and Snc1/2 facilitates CME initiation in growing daughter cells and directly links polarized CME to polarized secretion.

Contractile vacuole complexes (CVCs) are complex osmoregulatory organelles, with vesicular (bladder) and tubular (spongiome) subcompartments. The mechanisms that underlie their formation and maintenance within the eukaryotic endomembrane network are poorly understood. In the Ciliate Tetrahymena thermophila, six differentiated CORVETs (class C core vacuole/endosome tethering complexes), with Vps8 subunits designated A-F, are likely to direct endosomal trafficking. Vps8Dp localizes to both bladder and spongiome. We show by inducible knockdown that VPS8D is essential to CVC organization and function. VPS8D knockdown increased susceptibility to osmotic shock, tolerated in the wildtype but triggering irreversible lethal swelling in the mutant. The knockdown rapidly triggered contraction of the spongiome and lengthened the period of the bladder contractile cycle. More prolonged knockdown resulted in disassembly of both the spongiome and bladder, and dispersal of proteins associated with those compartments. In stressed cells where the normally singular bladder is replaced by numerous vesicles bearing bladder markers, Vps8Dp concentrated conspicuously at long-lived inter-vesicle contact sites, consistent with tethering activity. Similarly, Vps8Dp in cell-free preparations accumulated at junctions formed after vacuoles came into close contact. Also consistent with roles for Vps8Dp in tethering and/or fusion were the emergence in knockdown cells of multiple vacuole-related structures, replacing the single bladder. SynopsisIn the Ciliate Tetrahymena thermophila, VPS8D, which encodes a subunit of a non-conventional CORVET complex, is an essential determinant of the contractile vacuole complex (CVC). VPS8D knockdown results in retraction and dispersal of the spongiome, and disappearance of the bladder, reinforcing the view that CVCs arise from endosomal trafficking. Intermediate knockdown phenotypes and Vps8Dp localization support a role in homotypic tethering. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=188 SRC="FIGDIR/small/566071v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@b652c6org.highwire.dtl.DTLVardef@1f456f7org.highwire.dtl.DTLVardef@79ab8dorg.highwire.dtl.DTLVardef@1edbf74_HPS_FORMAT_FIGEXP M_FIG C_FIG

Signal transduction through the prolactin receptor (PRLR) is crucial in pancreatic {beta}-cell pro-liferation, impacting pancreatic homeostasis. PRLR-induced JAK/STAT signaling is dynamic, involving changes in spatial organization of signaling molecules. Thus, the spatial organization of PRLR could have strong implications on signaling output. Internalization has been shown and modeled in other signaling pathways but has not been considered in a mathematical model of PRLR signaling. Here, we use live-cell fluorescence imaging, reconstitution approaches, and fluorescence correlation spectroscopy (FCS) to inform a mathematical model of PRLR signaling. Internal PRLR localization is observed in primary pancreatic tissue and in an engineered PRLR expression system. Our imaging data indicate the presence of intracellular and plasma membrane-bound receptor populations. We use FCS to resolve the membrane-bound PRLR population. Based on our data, we include internalization dynamics within an ordinary differential equation (ODE) model of PRLR signaling. We employ the model to explore how the spatial heterogeneity of PRLR affects downstream signaling. We show that the model is more sensitive to PRLR trafficking rates and ability to promote signaling than to its initial spatial distribution. Our data underscore the versatility of a modeling-imaging framework to quantitatively understand signal transduction in and beyond {beta}-cells. Significance StatementO_LIProlactin receptor (PRLR) signal transduction impacts the growth and survival of insulin-secreting cells, making this pathway a target for building our understanding of pancreatic homeostasis and exploring potential diabetes therapeutics. C_LIO_LILive fluorescence imaging techniques applied within an engineered PRLR expression platform indicate PRLR localization patterns consistent with primary pancreatic tissue and the presence of two spatially distinct PRLR populations. These observations inform a predictive mathematical model of PRLR signaling. C_LIO_LIIntegrating experimental data tailored to computational approaches shapes our understanding of complex, multiscale systems such as signal transduction. A generalizable modeling-imaging framework enables the study of molecular dynamics beyond {beta}-cells. C_LI

Calcyphosine (CAPS) is a highly conserved but little explored calcium-binding protein that shows elevated expression in many forms of human cancer. Here we uncover a role for CAPS in spindle formation during mitosis. Our experiments suggest that CAPS is a microtubule-binding, spindle-associated protein that helps create the kinetochore fibers that bind and segregate chromosomes. Knockdown of CAPS causes a variety of defects during mitosis, including uncongressed chromosomes and multi-polar spindles, as well as high levels of apoptosis and a reduced mitotic index. We find that CAPS promotes microtubule bundling, both in vitro and in cells, and knockdown of CAPS leads to reduction of thick k-fibers in the mitotic spindle. The high level of CAPS observed in many forms of cancer suggests that CAPS may promote cell proliferation, but our results indicate that CAPS overexpression has little effect on the cell cycle. This suggests that the high level of CAPS expression may be a consequence of cancer, rather than a driving force for cell proliferation.

Spatial and temporal tracking of fluorescent proteins in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active fluorescent proteins (FPs) fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae. SIGNIFICANCE STATEMENT- Quantitative endocytic assays are required to characterize factors that regulate both ligand-dependent and constitutive endocytosis. - We optimize fluorogen-activating proteins (FAPs) technology for use as a live cell imaging probe in yeast that fluoresces in the far-red range for quantitative endocytosis assays. - The FAP tools and approaches generated will facilitate quantitative endocytic and protein recycling assays for yeast cell biologists.

Chemical and mechanical nuclear-cytoplasmic communication across the nuclear envelope (NE) is largely mediated by the nuclear pore complex (NPC) and the linker of nucleoskeleton and cytoskeleton (LINC) complex, respectively. While NPC and LINC complex assembly are functionally related, the mechanisms responsible for this relationship remain poorly understood. Here, we investigated how the luminal ATPases associated with various cellular activities (AAA+) protein torsinA promotes NPC and LINC complex assembly using fluorescence fluctuation spectroscopy (FFS), quantitative photobleaching analyses, and functional cellular assays. We report that torsinA controls LINC complex-dependent nuclear-cytoskeletal coupling as a soluble hexameric AAA+ protein and interphase NPC biogenesis as a membrane-associated helical polymer. These findings help resolve the conflicting models of torsinA function that were recently proposed based on in vitro structural studies. Our results will enable future studies of the role of defective nuclear-cytoplasmic communication in DYT1 dystonia and other diseases caused by mutations in torsinA.

In yeast and humans, the conserved DENN-domain (Differentially Expressed in Normal and Neoplastic tissue) protein Avl9 is thought to play roles in membrane traffic and secretion, but its precise function remains poorly defined. Since DENN-containing proteins are associated with Rab GTPase function, we sought to understand Avl9 function in the context of Rab regulation. Here, we show that Avl9 localizes to peripheral punctae that are consistent with secretory vesicles. Moreover, we demonstrate genetic interactions and co-localization between Avl9 and numerous Rabs in the secretory and endosomal pathways, suggesting a potential function at the interface of secretion and recycling. Consistent with this role, avl9{Delta} results in defective recycling of the endosomal cargo Snc1 but does not alter plasma membrane delivery of an endocytosis-defective Snc1EN- mutant, suggesting that Avl9 is not directly involved in secretory traffic from the TGN to the plasma membrane. The avl9{Delta} recycling defect is exacerbated by the additional loss of RCY1 or SNX4, but not VPS35. Each of these three genes contributes to a distinct endosomal recycling pathway, indicating that Avl9 acts in conjunction with multiple recycling pathways. Summary StatementIn this study, Rioux et al. describe a role for the DENN domain protein Avl9, previously thought to regulate secretion, as a novel factor involved in recycling of cargos from endosomal compartments.

During cytokinesis of animals and fungi, a contractile actomyosin ring (CAR) assembles at target locations and constricts to drive cell separation. In animal cells, the position of the CAR is determined by the mitotic spindle, so that the cleavage plane is perpendicular to the mitotic axis. However, in budding yeasts, the location of CAR assembly is specified by a cortical septin cytoskeleton that recruits CAR components to the neck. In the polymorphic fungus Aureobasidium pullulans, we show that septins assemble at mother-bud necks and predict the site of CAR assembly. Cells lacking septins stochastically failed to assemble CARs at a subset of bud necks. However, even cells lacking all four core septins were able to assemble CARs at 75% of bud necks. Our findings suggest the existence of a novel CAR positioning strategy that requires neither septin scaffolds nor nuclear/spindle cues to enable CAR assembly and constriction at bud necks. eTOC SUMMARYBudding yeasts are thought to use septins to mark mother-bud necks as sites for cytokinesis. Here, we find that the multibudding yeast Aureobasidium pullulans can position cytokinetic machinery at most bud necks even in the absence of septins, revealing a novel pathway to mark cytokinesis sites.

Microtubule dynamics change during cell division to enable rapid microtubule network remodeling. The switching from microtubule growth to shrinkage is attributed to the loss of a stabilizing GTP-cap structure at the growing microtubule end. The size of the GTP-cap is a result of a balance between GTP-tubulin addition to the microtubule end and subsequent GTP-hydrolysis in the microtubule lattice. Whether the cell-cycle-dependent changes in microtubule dynamics are supported by concurrent modulation of the stabilizing GTP-cap size is not known. Here, we use high spatiotemporal resolution live-cell imaging of EB1, an established marker for the GTP-cap, to directly determine the relationship between GTP-cap size and microtubule growth rate throughout the cell cycle. Our data reveal that GTP-cap size for matching growth rates is reduced during mitosis. Comparison of EB1 comets on astral versus spindle microtubules reveals that the scaling between the GTP-cap size and microtubule growth rate is not spatially regulated in mitosis. We find that these regulatory patterns are conserved across epithelial cells from two different species. Taken together, our findings reveal modulation of GTP-cap size as a cell-cycle-regulated mechanism for tuning microtubule stability. Significance StatementMicrotubule dynamics are altered during the cell cycle to enable rapid microtubule network remodeling and accurate chromosome segregation. By comparing EB1 comets on microtubule ends during different cell cycle stages, the authors find that microtubule GTP-cap size is subject to global differential regulation during specific cell cycle stages. These results identify modulation of microtubule stabilizing GTP-cap size as a previously underappreciated, cell-cycle-regulated mechanism for tuning microtubule stability throughout the cell cycle.

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae. Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

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.

The contractile vacuole complex (CVC) is a dynamic and morphologically complex membrane organelle, comprised of a large vesicle (bladder) linked with a tubular reticulum (spongiome). CVCs provide key osmoregulatory roles across diverse eukaryotic lineages, but probing the mechanisms underlying the structure and function is hampered by the limited tools available for in vivo analysis. In the experimentally tractable ciliate Tetrahymena thermophila, we describe four proteins that, as endogenously tagged constructs, localize specifically to distinct CVC zones. The DOPEY homolog Dop1p and the CORVET subunit Vps8Dp localize both to the bladder and spongiome but with different local distributions that are sensitive to osmotic perturbation, while the lipid scramblase Scr7p co-localizes with Vps8Dp. The H+- ATPase subunit Vma4 is spongiome-specific. The live imaging permitted by these probes revealed dynamics at multiple scales including rapid exchange of CVC-localized and soluble protein pools vs. lateral diffusion in the spongiome, spongiome extension and branching, and CVC formation during mitosis. While the association with DOP1 and VPS8D implicate the CVC in endosomal trafficking, both the bladder and spongiome are isolated from bulk endocytic input. Summary statementIn the ciliate Tetrahymena thermophila, four proteins are shown to provide markers for different zones of the contractile vacuole complex. They shed light on its formation and maintenance by enabling in vivo analysis of its dynamics.

Although cold temperatures have long been used to depolymerize microtubules, how temperature specifically affects the polymerization and depolymerization activities of tubulin proteins and how these lead to changes in microtubule networks in cells has not been established. We investigated these questions in budding yeast, an organism found in diverse environments and therefore predicted to exhibit dynamic microtubules across a broad range of temperatures. We measured the dynamics of GFP-labeled microtubules in living cells and found that lowering the temperature from 37{degrees}C to 10{degrees}C decreased the rates of both polymerization and depolymerization, decreased the amount of polymer assembled before catastrophes and decreased the frequency of microtubule emergence from nucleation sites. Lowering to 4{degrees}C caused rapid loss of almost all microtubule polymer. We provide evidence that these effects on microtubule dynamics may be explained in part by changes in the co-factor-dependent conformational dynamics of tubulin proteins. Ablation of tubulin-binding co-factors further sensitizes cells and their microtubules to low temperatures, and we highlight a specific role for TBCB/Alf1 in microtubule maintenance at low temperatures. Finally, we show that inhibiting the maturation cycle of tubulin by using a point mutant in {beta}-tubulin confers hyper-stable microtubules at low temperatures, rescues the requirement for TBCB/Alf1, and improves the cold tolerance of the yeast. Together, these results reveal an unappreciated step in the tubulin cycle in cells and suggest that this step may be a key limiting factor in the thermal tolerance of organisms.

How cells regulate the supply of - and {beta}-tubulin monomers to meet the demand for {beta}- heterodimers while avoiding consequences of monomer imbalance is not understood. We investigate the role of gene copy number in tubulin regulation and how shifting the expression of - or {beta}-tubulin genes impacts tubulin proteostasis and microtubule function. We find that - tubulin gene copy number is important for maintaining an excess -tubulin protein compared to {beta}-tubulin protein and preventing accumulation of super-stoichiometric {beta}-tubulin. Super- stoichiometric {beta}-tubulin is toxic to cells, leading to loss of microtubules, formation of non- microtubule assemblies of tubulin, and disrupted cell proliferation. In contrast, decreased {beta}- tubulin or increased -tubulin has minor effects. We provide evidence that cells rapidly equilibrate the concentration of -tubulin protein during shifts in -tubulin isotype expression to maintain a ratio in excess of {beta}-tubulin. We propose an asymmetric relationship between - and {beta}-tubulins, where -tubulins are maintained in excess to supply {beta}-heterodimers and limit the accumulation of {beta}-tubulin monomers.

Septin filaments build structures such as rings, lattices and gauzes that serve as platforms for localizing signaling and organizing cell membranes. How cells control the geometry of septin assemblies in poorly understood. We show here that septins are isodesmic polymers, in contrast to cooperative polymerization exhibited by F-actin and microtubules. We constructed a physical model to analyze and interpret how septin assemblies change in the presence of regulators in yeast extracts. Notably filaments differ in length and curvature in yeast extract compared to pure protein indicating cellular regulators modulate intrinsic biophysical features. Combining analysis of extracts from regulatory mutants with simulations, we found increased filament flexibility and reduced filament fragmentation promote assembly of septin rings, whereas reduced flexibility in crowded environments promotes local filament alignment. This work demonstrates how tuning of intrinsic features of septin filament assembly by regulatory proteins yields a diverse array of structures observed in cells.

Primary cilia are sensory cellular organelles crucial for organ development and homeostasis. Ciliogenesis in polarized epithelial cells requires Rab19-mediated clearing of apical cortical actin to allow the cilium to grow from the apically-docked basal body into the extracellular space. Loss of the lysosomal membrane-tethering HOPS complex disrupts this actin-clearing and ciliogenesis, but it remains unclear how ciliary function of HOPS relates to its canonical function in regulating late endosome-lysosome fusion. Here, we show that disruption of HOPS-dependent lysosomal fusion indirectly impairs actin-clearing and ciliogenesis by disrupting the targeting of Rab19 to the basal body. We also find that Rab19 functions in endolysosomal cargo trafficking apart from its previously-identified role in ciliogenesis. In summary, we show that inhibition of lysosomal fusion abnormally accumulates Rab19 on late endosomes, thus depleting Rab19 from the basal body and thereby disrupting Rab19-mediated actin-clearing and ciliogenesis. Summary statementLoss of HOPS-mediated lysosomal fusion indirectly blocks apical actin clearing and ciliogenesis in polarized epithelia by trapping Rab19 on late endosomes and depleting Rab19 from the basal body.

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.