Vinculin Is Part of the Cadherin–Catenin Junctional Complex: Complex Formation between α-Catenin and Vinculin

In epithelial cells, α-, β-, and γ-catenin are involved in linking the peripheral microfilament belt to the transmembrane protein E-cadherin. α-Catenin exhibits sequence homologies over three regions to vinculin, another adherens junction protein. While vinculin is found in cell–matrix and cell–cell contacts, α-catenin is restricted to the latter. To elucidate, whether vinculin is part of the cell–cell junctional complex, we investigated complex formation and intracellular targeting of vinculin and α-catenin. We show that α-catenin colocalizes at cell–cell contacts with endogenous vinculin and also with the transfected vinculin head domain forming immunoprecipitable complexes. In vitro, the vinculin NH₂-terminal head binds to α-catenin, as seen by immunoprecipitation, dot overlay, cosedimentation, and surface plasmon resonance measurements. The K_d of the complex was determined to 2–4 × 10⁻⁷ M. As seen by overlays and affinity mass spectrometry, the COOH-terminal region of α-catenin is involved in this interaction.     

NH2-terminal Deletion of β-Catenin Results in Stable Colocalization of Mutant β-Catenin with Adenomatous Polyposis Coli Protein and Altered MDCK Cell Adhesion

β-Catenin is essential for the function of cadherins, a family of Ca²⁺-dependent cell–cell adhesion molecules, by linking them to α-catenin and the actin cytoskeleton. β-Catenin also binds to adenomatous polyposis coli (APC) protein, a cytosolic protein that is the product of a tumor suppressor gene mutated in colorectal adenomas. We have expressed mutant β-catenins in MDCK epithelial cells to gain insights into the regulation of β-catenin distribution between cadherin and APC protein complexes and the functions of these complexes. Full-length β-catenin, β-catenin mutant proteins with NH₂-terminal deletions before (ΔN90) or after (ΔN131, ΔN151) the α-catenin binding site, or a mutant β-catenin with a COOH-terminal deletion (ΔC) were expressed in MDCK cells under the control of the tetracycline-repressible transactivator. All β-catenin mutant proteins form complexes and colocalize with E-cadherin at cell–cell contacts; ΔN90, but neither ΔN131 nor ΔN151, bind α-catenin. However, β-catenin mutant proteins containing NH₂-terminal deletions also colocalize prominently with APC protein in clusters at the tips of plasma membrane protrusions; in contrast, full-length and COOH-terminal– deleted β-catenin poorly colocalize with APC protein. NH₂-terminal deletions result in increased stability of β-catenin bound to APC protein and E-cadherin, compared with full-length β-catenin. At low density, MDCK cells expressing NH₂-terminal–deleted β-catenin mutants are dispersed, more fibroblastic in morphology, and less efficient in forming colonies than parental MDCK cells. These results show that the NH₂ terminus, but not the COOH terminus of β-catenin, regulates the dynamics of β-catenin binding to APC protein and E-cadherin. Changes in β-catenin binding to cadherin or APC protein, and the ensuing effects on cell morphology and adhesion, are independent of β-catenin binding to α-catenin. These results demonstrate that regulation of β-catenin binding to E-cadherin and APC protein is important in controlling epithelial cell adhesion.     

Antagonism of Cell Adhesion by an α-Catenin Mutant, and of the Wnt-signaling Pathway by α-Catenin in Xenopus Embryos

In Xenopus laevis development, β-catenin plays an important role in the Wnt-signaling pathway by establishing the Nieuwkoop center, which in turn leads to specification of the dorsoventral axis. Cadherins are essential for embryonic morphogenesis since they mediate calcium-dependent cell–cell adhesion and can modulate β-catenin signaling. α-catenin links β-catenin to the actin-based cytoskeleton. To study the role of endogenous α-catenin in early development, we have made deletion mutants of αN-catenin. The binding domain of β-catenin has been mapped to the NH₂-terminal 210 amino acids of αN-catenin. Overexpression of mutants lacking the COOH-terminal 230 amino acids causes severe developmental defects that reflect impaired calcium-dependent blastomere adhesion. Lack of normal adhesive interactions results in a loss of the blastocoel in early embryos and ripping of the ectodermal layer during gastrulation. The phenotypes of the dominant-negative mutants can be rescued by coexpressing full-length αN-catenin or a mutant of β-catenin that lacks the internal armadillo repeats.

We next show that coexpression of αN-catenin antagonizes the dorsalizing effects of β-catenin and Xwnt-8. This can be seen phenotypically, or by studying the effects of expression on the downstream homeobox gene Siamois. Thus, α-catenin is essential for proper morphogenesis of the embryo and may act as a regulator of the intracellular β-catenin signaling pathway in vivo.

     

α–E-catenin binds to dynamitin and regulates dynactin-mediated intracellular traffic

α–Epithelial catenin (E-catenin) is an important cell–cell adhesion protein. In this study, we show that α–E-catenin also regulates intracellular traffic by binding to the dynactin complex component dynamitin. Dynactin-mediated organelle trafficking is increased in α–E-catenin^−/− keratinocytes, an effect that is reversed by expression of exogenous α–E-catenin. Disruption of adherens junctions in low-calcium media does not affect dynactin-mediated traffic, indicating that α–E-catenin regulates traffic independently from its function in cell–cell adhesion. Although neither the integrity of dynactin–dynein complexes nor their association with vesicles is affected by α–E-catenin, α–E-catenin is necessary for the attenuation of microtubule-dependent trafficking by the actin cytoskeleton. Because the actin-binding domain of α–E-catenin is necessary for this regulation, we hypothesize that α–E-catenin functions as a dynamic link between the dynactin complex and actin and, thus, integrates the microtubule and actin cytoskeleton during intracellular trafficking.     

Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

Protein Kinase C Activation Upregulates Intercellular Adhesion of α-Catenin–negative Human Colon Cancer Cell Variants via Induction of Desmosomes

The α-catenin molecule links E-cadherin/ β-catenin or E-cadherin/plakoglobin complexes to the actin cytoskeleton. We studied several invasive human colon carcinoma cell lines lacking α-catenin. They showed a solitary and rounded morphotype that correlated with increased invasiveness. These round cell variants acquired a more normal epithelial phenotype upon transfection with an α-catenin expression plasmid, but also upon treatment with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Video registrations showed that the cells started to establish elaborated intercellular junctions within 30 min after addition of TPA. Interestingly, this normalizing TPA effect was not associated with α-catenin induction. Classical and confocal immunofluorescence showed only minor TPA-induced changes in E-cadherin staining. In contrast, desmosomal and tight junctional proteins were dramatically rearranged, with a conversion from cytoplasmic clusters to obvious concentration at cell–cell contacts and exposition at the exterior cell surface. Electron microscopical observations revealed the TPA-induced appearance of typical desmosomal plaques. TPA-restored cell–cell adhesion was E-cadherin dependent as demonstrated by a blocking antibody in a cell aggregation assay. Addition of an antibody against the extracellular part of desmoglein-2 blocked the TPA effect, too. Remarkably, the combination of anti–E-cadherin and anti-desmoglein antibodies synergistically inhibited the TPA effect.

Our studies show that it is possible to bypass the need for normal α-catenin expression to establish tight intercellular adhesion by epithelial cells. Apparently, the underlying mechanism comprises upregulation of desmosomes and tight junctions by activation of the PKC signaling pathway, whereas E-cadherin remains essential for basic cell–cell adhesion, even in the absence of α-catenin.

     

Involvement of ZO-1 in Cadherin-based Cell Adhesion through Its Direct Binding to α Catenin and Actin Filaments

ZO-1, a 220-kD peripheral membrane protein consisting of an amino-terminal half discs large (dlg)-like domain and a carboxyl-terminal half domain, is concentrated at the cadherin-based cell adhesion sites in non-epithelial cells. We introduced cDNAs encoding the full-length ZO-1, its amino-terminal half (N-ZO-1), and carboxyl-terminal half (C-ZO-1) into mouse L fibroblasts expressing exogenous E-cadherin (EL cells). The full-length ZO-1 as well as N-ZO-1 were concentrated at cadherin-based cell–cell adhesion sites. In good agreement with these observations, N-ZO-1 was specifically coimmunoprecipitated from EL transfectants expressing N-ZO-1 (NZ-EL cells) with the E-cadherin/α, β catenin complex. In contrast, C-ZO-1 was localized along actin stress fibers. To examine the molecular basis of the behavior of these truncated ZO-1 molecules, N-ZO-1 and C-ZO-1 were produced in insect Sf9 cells by recombinant baculovirus infection, and their direct binding ability to the cadherin/catenin complex and the actin-based cytoskeleton, respectively, were examined in vitro. Recombinant N-ZO-1 bound directly to the glutathione-S-transferase fusion protein with α catenin, but not to that with β catenin or the cytoplasmic domain of E-cadherin. The dissociation constant between N-ZO-1 and α catenin was ~0.5 nM. On the other hand, recombinant C-ZO-1 was specifically cosedimented with actin filaments in vitro with a dissociation constant of ~10 nM. Finally, we compared the cadherin-based cell adhesion activity of NZ-EL cells with that of parent EL cells. Cell aggregation assay revealed no significant differences among these cells, but the cadherin-dependent intercellular motility, i.e., the cell movement in a confluent monolayer, was significantly suppressed in NZ-EL cells. We conclude that in nonepithelial cells, ZO-1 works as a cross-linker between cadherin/catenin complex and the actin-based cytoskeleton through direct interaction with α catenin and actin filaments at its amino- and carboxyl-terminal halves, respectively, and that ZO-1 is a functional component in the cadherin-based cell adhesion system.     

Direct interaction of β‐catenin with nuclear ESM1 supports stemness of metastatic prostate cancer

Wnt/β‐catenin signaling is frequently activated in advanced prostate cancer and contributes to therapy resistance and metastasis. However, activating mutations in the Wnt/β‐catenin pathway are not common in prostate cancer, suggesting alternative regulations may exist. Here, we report that the expression of endothelial cell‐specific molecule 1 (ESM1), a secretory proteoglycan, is positively associated with prostate cancer stemness and progression by promoting Wnt/β‐catenin signaling. Elevated ESM1 expression correlates with poor overall survival and metastasis. Accumulation of nuclear ESM1, instead of cytosolic or secretory ESM1, supports prostate cancer stemness by interacting with the ARM domain of β‐catenin to stabilize β‐catenin–TCF4 complex and facilitate the transactivation of Wnt/β‐catenin signaling targets. Accordingly, activated β‐catenin in turn mediates the nuclear entry of ESM1. Our results establish the significance of mislocalized ESM1 in driving metastasis in prostate cancer by coordinating the Wnt/β‐catenin pathway, with implications for its potential use as a diagnostic or prognostic biomarker and as a candidate therapeutic target in prostate cancer.     

Effects of Forced Expression of an NH2-terminal Truncated β-Catenin on Mouse Intestinal Epithelial Homeostasis

β-Catenin functions as a downstream component of the Wnt/Wingless signal transduction pathway and as an effector of cell–cell adhesion through its association with cadherins. To explore the in vivo effects of β-catenin on proliferation, cell fate specification, adhesion, and migration in a mammalian epithelium, a human NH₂-terminal truncation mutant (ΔN89β-catenin) was expressed in the 129/Sv embryonic stem cell–derived component of the small intestine of adult C57Bl/6–ROSA26↔ 129/Sv chimeric mice. ΔN89β-Catenin was chosen because mutants of this type are more stable than the wild-type protein, and phenocopy activation of the Wnt/Wingless signaling pathway in Xenopus and Drosophila. ΔN89β-Catenin had several effects. Cell division was stimulated fourfold in undifferentiated cells located in the proliferative compartment of the intestine (crypts of Lieberkühn). The proliferative response was not associated with any discernible changes in cell fate specification but was accompanied by a three- to fourfold increase in crypt apoptosis. There was a marked augmentation of E-cadherin at the adherens junctions and basolateral surfaces of 129/Sv (ΔN89β-catenin) intestinal epithelial cells and an accompanying slowing of cellular migration along crypt-villus units. 1–2% of 129/Sv (ΔN89β-catenin) villi exhibited an abnormal branched architecture. Forced expression of ΔN89β-catenin expression did not perturb the level or intracellular distribution of the tumor suppressor adenomatous polyposis coli (APC). The ability of ΔN89β-catenin to interact with normal cellular pools of APC and/or augmented pools of E-cadherin may have helped prevent the 129/Sv gut epithelium from undergoing neoplastic transformation during the 10-mo period that animals were studied. Together, these in vivo studies emphasize the importance of β-catenin in regulating normal adhesive and signaling functions within this epithelium.     

Signaling and Adhesion Activities of Mammalian β-Catenin and Plakoglobin in Drosophila

The armadillo protein of Drosophila and its vertebrate homologues, β-catenin and plakoglobin, are implicated in cell adhesion and wnt signaling. Here, we examine the conservation of these two functions by assaying the activities of mammalian β-catenin and plakoglobin in Drosophila. We show that, in the female germ line, both mammalian β-catenin and plakoglobin complement an armadillo mutation. We also show that shotgun mutant germ cells (which lack Drosophila E-cadherin) have a phenotype identical to that of armadillo mutant germ cells. It therefore appears that armadillo's role in the germ line is solely in a complex with Drosophila E-cadherin (possibly an adhesion complex), and both β-catenin and plakoglobin can function in Drosophila cadherin complexes. In embryonic signaling assays, we find that plakoglobin has no detectable activity whereas β-catenin's activity is weak. Surprisingly, when overexpressed, either in embryos or in wing imaginal disks, both β-catenin and plakoglobin have dominant negative activity on signaling, an effect also obtained with COOH-terminally truncated armadillo. We suggest that the signaling complex, which has been shown by others to comprise armadillo and a member of the lymphocyte enhancer binding factor-1/T cell factor–family, may contain an additional factor that normally binds to the COOH-terminal region of armadillo.     

Cleavage of β-Catenin and Plakoglobin and Shedding of VE-Cadherin during Endothelial Apoptosis: Evidence for a Role for Caspases and Metalloproteinases

Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell–matrix and cell–cell contacts. In this study, we show that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. β-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding α-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved β-catenin from lysates of apoptotic cells does not bind to endogenous α-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell–cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this “shedding” of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of β-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival.     

β-Cells retain a pool of insulin-containing secretory vesicles regulated by adherens junctions and the cadherin-binding protein p120 catenin

The β-cells of the islets of Langerhans are the sole producers of insulin in the human body. In response to rising glucose levels, insulin-containing vesicles inside β-cells fuse with the plasma membrane and release their cargo. However, the mechanisms regulating this process are only partly understood. Previous evidence indicated reductions in α-catenin elevate insulin release, while reductions in β-catenin decrease insulin release. α- and β-catenin contribute to cellular regulation in a range of ways but one is as members of the adherens junction complex. Therefore, we investigated the effects of adherens junctions on insulin release. We show in INS-1E β-cells knockdown of either E- or N-cadherin had only small effects on insulin secretion, but simultaneous knockdown of both cadherins resulted in a significant increase in basal insulin release to the same level as glucose-stimulated release. This double knockdown also significantly attenuated levels of p120 catenin, a cadherin-binding partner involved in regulating cadherin turnover. Conversely, reducing p120 catenin levels with siRNA destabilized both E- and N-cadherin, and this was also associated with an increase in levels of insulin secreted from INS-1E cells. Furthermore, there were also changes in these cells consistent with higher insulin release, namely reductions in levels of F-actin and increased intracellular free Ca²⁺ levels in response to KCl-induced membrane depolarization. Taken together, these data provide evidence that adherens junctions play important roles in retaining a pool of insulin secretory vesicles within the cell and establish a role for p120 catenin in regulating this process.     

Dynamics of β-Catenin Interactions with APC Protein Regulate Epithelial Tubulogenesis

Epithelial tubulogenesis involves complex cell rearrangements that require control of both cell adhesion and migration, but the molecular mechanisms regulating these processes during tubule development are not well understood. Interactions of the cytoplasmic protein, β-catenin, with several molecular partners have been shown to be important for cell signaling and cell–cell adhesion. To examine if β-catenin has a role in tubulogenesis, we tested the effect of expressing NH₂-terminal deleted β-catenins in an MDCK epithelial cell model for tubulogenesis. After one day of treatment, hepatocyte growth factor/scatter factor (HGF/ SF)-stimulated MDCK cysts initiated tubulogenesis by forming many long cell extensions. Expression of NH₂-terminal deleted β-catenins inhibited formation of these cell extensions. Both ΔN90 β-catenin, which binds to α-catenin, and ΔN131 β-catenin, which does not bind to α-catenin, inhibited formation of cell extensions and tubule development, indicating that a function of β-catenin distinct from its role in cadherin-mediated cell–cell adhesion is important for tubulogenesis. In cell extensions from parental cysts, adenomatous polyposis coli (APC) protein was localized in linear arrays and in punctate clusters at the tips of extensions. Inhibition of cell extension formation correlated with the colocalization and accumulation of NH₂-terminal deleted β-catenin in APC protein clusters and the absence of linear arrays of APC protein. Continued HGF/ SF treatment of parental cell MDCK cysts resulted in cell proliferation and reorganization of cell extensions into multicellular tubules. Similar HGF/SF treatment of cysts derived from cells expressing NH₂-terminal deleted β-catenins resulted in cells that proliferated but formed cell aggregates (polyps) within the cyst rather than tubules. Our results demonstrate an unexpected role for β-catenin in cell migration and indicate that dynamic β-catenin–APC protein interactions are critical for regulating cell migration during epithelial tubulogenesis.     

p120-catenin is a key component of the cadherin-gamma-secretase supercomplex

In this work, we show several previously unknown features of p120-catenin in a cadherin–catenin complex that are critical for our understanding of cadherin-based adhesion and signaling. We show that in human epithelial A-431 cells, nearly all p120 molecules engage in high-affinity interaction with E-cadherin–catenin complexes located at the cellular surface. p120 is positioned in proximity to α-catenin in the complex with cadherin. These findings suggest a functional cooperation between p120 and α-catenin in cadherin-based adhesion. A low level of cadherin-free p120 molecules, in contrast, could facilitate p120-dependent signaling. Finally, we present compelling evidence that p120 is a key linker cementing the E-cadherin–catenin complex with the transmembrane protease γ-secretase. The cell–cell contact location of this supercomplex makes it an important candidate for conducting different signals that rely on γ-secretase proteolytic activity.     

E-cadherin phosphorylation occurs during its biosynthesis to promote its cell surface stability and adhesion

E-cadherin is highly phosphorylated within its β-catenin–binding region, and this phosphorylation increases its affinity for β-catenin in vitro. However, the identification of key serines responsible for most cadherin phosphorylation and the adhesive consequences of modification at such serines have remained unknown. In this study, we show that as few as three serines in the β-catenin–binding domain of E-cadherin are responsible for most radioactive phosphate incorporation. These serines are required for binding to β-catenin and the mutual stability of both E-cadherin and β-catenin. Cells expressing a phosphodeficient (3S>A) E-cadherin exhibit minimal cell–cell adhesion due to enhanced endocytosis and degradation through a lysosomal compartment. Conversely, negative charge substitution at these serines (3S>D) antagonizes cadherin endocytosis and restores wild-type levels of adhesion. The cadherin kinase is membrane proximal and modifies the cadherin before it reaches the cell surface. Together these data suggest that E-cadherin phosphorylation is largely constitutive and integral to cadherin–catenin complex formation, surface stability, and function.     

Centrosomal enrichment and proteasomal degradation of SYS-1/β-catenin requires the microtubule motor dynein

The Caenorhabditis elegans Wnt/β-catenin asymmetry (WβA) pathway utilizes asymmetric regulation of SYS-1/β-catenin and POP-1/TCF coactivators. WβA differentially regulates gene expression during cell fate decisions, specifically by asymmetric localization of determinants in mother cells to produce daughters biased toward their appropriate cell fate. Despite the induction of asymmetry, β-catenin localizes symmetrically to mitotic centrosomes in both mammals and C. elegans. Owing to the mitosis-specific localization of SYS-1 to centrosomes and enrichment of SYS-1 at kinetochore microtubules when SYS-1 centrosomal loading is disrupted, we investigated active trafficking in SYS-1 centrosomal localization. Here, we demonstrate that trafficking by microtubule motor dynein is required to maintain SYS-1 centrosomal enrichment, by dynein RNA interference (RNAi)-mediated decreases in SYS-1 centrosomal enrichment and by temperature-sensitive allele of the dynein heavy chain. Conversely, we observe depletion of microtubules by nocodazole treatment or RNAi of dynein-proteasome adapter ECPS-1 exhibits increased centrosomal enrichment of SYS-1. Moreover, disruptions to SYS-1 or negative regulator microtubule trafficking are sufficient to significantly exacerbate SYS-1 dependent cell fate misspecifications. We propose a model whereby retrograde microtubule-mediated trafficking enables SYS-1 enrichment at centrosomes, enhancing its eventual proteasomal degradation. These studies support the link between centrosomal localization and enhancement of proteasomal degradation, particularly for proteins not generally considered “centrosomal.”     

Glucose regulates expression of pro-inflammatory genes,IL-1β and IL-12, through a mechanism involving hexosamine biosynthesis pathway-dependent regulation of α-E catenin

High glucose levels are associated with changes in macrophage polarisation and evidence indicates that the sustained or even short-term high glucose levels modulate inflammatory responses in macrophages. However, the mechanism by which macrophages can sense the changes in glucose levels are not clearly understood. We find that high glucose levels rapidly increase the α-E catenin protein level in RAW264.7 macrophages. We also find an attenuation of glucose-induced increase in α-E catenin when hexosamine biosynthesis (HB) pathway is inhibited either with glutamine depletion or with the drugs azaserine and tunicamycin. This indicates the involvement of HB pathway in this process. Then, we investigated the potential role of α-E catenin in glucose-induced macrophage polarisation. We find that the reduction in α-E catenin level using siRNA attenuates the glucose-induced changes of both IL-1β and IL-12 mRNA levels under LPS-stimulated condition but does not affect TNF-α expression. Together this indicates that α-E catenin can sense the changes in glucose levels in macrophages via HB pathway and also can modulate the glucose-induced gene expression of inflammatory markers such as IL-1β and IL-12. This identifies a new part of the mechanism by which macrophages are able to respond to changes in glucose levels.     

Microbial Conversion of α-Ionone, α-Methylionone, and α-Isomethylionone

α-Ionone, α-methylionone, and α-isomethylionone were converted by Aspergillus niger JTS 191. The individual bioconversion products from α-ionone were isolated and identified by spectrometry and organic synthesis. The major products were cis-3-hydroxy-α-ionone, trans-3-hydroxy-α-ionone, and 3-oxo-α-ionone. 2,3-Dehydro-α-ionone, 3,4-dehydro-β-ionone, and 1-(6,6-dimethyl-2-methylene-3-cyclohexenyl)-buten-3-one were also identified. Analogous bioconversion products from α-methylionone and α-isomethylionone were also identified. From results of gas-liquid chromatographic analysis during the fermentation, we propose a metabolic pathway for α-ionones and elucidation of stereochemical features of the bioconversion.