Nucleotide Sequence of the F(1)-ATPase alpha Subunit Gene from Maize Mitochondria

The α subunit of the F₁-ATPase complex of maize is a mitochondrial translational product, presumably encoded by the mitochondrial genome. Based on nucleotide and amino acid homology, we have identified a mitochondrial gene, designated atpα, that appears to code for the F₁-ATPase α subunit of Zea mays. The atpα gene is present as a single copy in the maize. Texas cytoplasm and is actively transcribed. The maize α polypeptide has a predicted length of 508 amino acids and a molecular mass of 55,187 daltons. Amino acid homologies between the maize mitochondrial α subunit and the tobacco chloroplast CF₁ and Escherichia coli α subunits are 54 and 51%, respectively. The origin of the atpα gene is discussed.     

Plasma membrane aminoglycerolipid flippase function is required for signaling competence in the yeast mating pheromone response pathway

The class 4 P-type ATPases (“flippases”) maintain membrane asymmetry by translocating phosphatidylethanolamine and phosphatidylserine from the outer leaflet to the cytosolic leaflet of the plasma membrane. In Saccharomyces cerevisiae, five related gene products (Dnf1, Dnf2, Dnf3, Drs2, and Neo1) are implicated in flipping of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine. In MATa cells responding to α-factor, we found that Dnf1, Dnf2, and Dnf3, as well as the flippase-activating protein kinase Fpk1, localize at the projection (“shmoo”) tip where polarized growth is occurring and where Ste5 (the central scaffold protein of the pheromone-initiated MAPK cascade) is recruited. Although viable, a MATa dnf1∆ dnf2∆ dnf3∆ triple mutant exhibited a marked decrease in its ability to respond to α-factor, which we could attribute to pronounced reduction in Ste5 stability resulting from an elevated rate of its Cln2⋅Cdc28-initiated degradation. Similarly, a MATa dnf1∆ dnf3∆ drs2∆ triple mutant also displayed marked reduction in its ability to respond to α-factor, which we could attribute to inefficient recruitment of Ste5 to the plasma membrane due to severe mislocalization of the cellular phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate pools. Thus proper remodeling of plasma membrane aminoglycerolipids and phosphoinositides is necessary for efficient recruitment, stability, and function of the pheromone signaling apparatus.     

Cell surface changes in the Candida albicans mitochondrial mutant goa1Δ are associated with reduced recognition by innate immune cells

We have previously characterized several fungal‐specific proteins from the human pathogen Candida albicans that either encode subunits of mitochondria Complex I (CI) of the electron transport chain (ETC) or regulate CI activity (Goa1p). Herein, the role of energy production and cell wall gene expression is investigated in the mitochondria mutant goa1Δ. We show that downregulation of cell wall‐encoding genes in the goa1Δ results in sensitivity to cell wall inhibitors such as Congo red and Calcofluor white, reduced phagocytosis by a macrophage cell line, reduced recognition by macrophage receptors, and decreased expression of cytokines such as IL‐6, IL‐10 and IFN‐γ. In spite of the reduced recognition by macrophages, the goa1Δ is still killed to the same extent as control strains. We also demonstrate that expression of the epithelial cell receptors E‐cadherin and EGFR is also reduced in the presence of goa1Δ. Together, our data demonstrate the importance of mitochondria in the expression of cell wall biomolecules and the interaction of C. albicans with innate immune and epithelial cells. Our underlying premise is thatmitochondrial proteins such as Goa1p and other fungal‐specific mitochondrial proteins regulate critical functions in cell growth and in virulence. As such, they remain as valid drug targets for antifungal drug discovery.     

Neurons and Glia Modify Receptor Protein-tyrosine Phosphatase ζ (RPTPζ)/Phosphacan with Cell-specific O-Mannosyl Glycans in the Developing Brain

Protein O-mannosylation is a glycan modification that is required for normal nervous system development and function. Mutations in genes involved in protein O-mannosyl glycosylation give rise to a group of neurodevelopmental disorders known as congenital muscular dystrophies (CMDs) with associated CNS abnormalities. Our previous work demonstrated that receptor protein-tyrosine phosphatase ζ (RPTPζ)/phosphacan is hypoglycosylated in a mouse model of one of these CMDs, known as muscle-eye-brain disease, a disorder that is caused by loss of an enzyme (protein O-mannose β-1,2-N-acetylglucosaminyltransferase 1) that modifies O-mannosyl glycans. In addition, monoclonal antibodies Cat-315 and 3F8 were demonstrated to detect O-mannosyl glycan modifications on RPTPζ/phosphacan. Here, we show that O-mannosyl glycan epitopes recognized by these antibodies define biochemically distinct glycoforms of RPTPζ/phosphacan and that these glycoforms differentially decorate the surface of distinct populations of neural cells. To provide a further structural basis for immunochemically based glycoform differences, we characterized the O-linked glycan heterogeneity of RPTPζ/phosphacan in the early postnatal mouse brain by multidimensional mass spectrometry. Structural characterization of the O-linked glycans released from purified RPTPζ/phosphacan demonstrated that this protein is a significant substrate for protein O-mannosylation and led to the identification of several novel O-mannose-linked glycan structures, including sulfo-N-acetyllactosamine containing modifications. Taken together, our results suggest that specific glycan modifications may tailor the function of this protein to the unique needs of specific cells. Furthermore, their absence in CMDs suggests that hypoglycosylation of RPTPζ/phosphacan may have different functional consequences in neurons and glia.     

Antagonistic Regulation of Parvalbumin Expression and Mitochondrial Calcium Handling Capacity in Renal Epithelial Cells

Parvalbumin (PV) is a cytosolic Ca²⁺-binding protein acting as a slow-onset Ca²⁺ buffer modulating the shape of Ca²⁺ transients in fast-twitch muscles and a subpopulation of neurons. PV is also expressed in non-excitable cells including distal convoluted tubule (DCT) cells of the kidney, where it might act as an intracellular Ca²⁺ shuttle facilitating transcellular Ca²⁺ resorption. In excitable cells, upregulation of mitochondria in “PV-ergic” cells in PV-/- mice appears to be a general hallmark, evidenced in fast-twitch muscles and cerebellar Purkinje cells. Using Gene Chip Arrays and qRT-PCR, we identified differentially expressed genes in the DCT of PV-/- mice. With a focus on genes implicated in mitochondrial Ca²⁺ transport and membrane potential, uncoupling protein 2 (Ucp2), mitocalcin (Efhd1), mitochondrial calcium uptake 1 (Micu1), mitochondrial calcium uniporter (Mcu), mitochondrial calcium uniporter regulator 1 (Mcur1), cytochrome c oxidase subunit 1 (COX1), and ATP synthase subunit β (Atp5b) were found to be up-upregulated. At the protein level, COX1 was increased by 31 ± 7%, while ATP-synthase subunit β was unchanged. This suggested that these mitochondria were better suited to uphold the electrochemical potential across the mitochondrial membrane, necessary for mitochondrial Ca²⁺ uptake. Ectopic expression of PV in PV-negative Madin-Darby canine kidney (MDCK) cells decreased COX1 and concomitantly mitochondrial volume, while ATP synthase subunit β levels remained unaffected. Suppression of PV by shRNA in PV-expressing MDCK cells led subsequently to an increase in COX1 expression. The collapsing of the mitochondrial membrane potential by the uncoupler CCCP occurred at lower concentrations in PV-expressing MDCK cells than in control cells. In support, a reduction of the relative mitochondrial mass was observed in PV-expressing MDCK cells. Deregulation of the cytoplasmic Ca²⁺ buffer PV in kidney cells was counterbalanced in vivo and in vitro by adjusting the relative mitochondrial volume and modifying the mitochondrial protein composition conceivably to increase their Ca²⁺-buffering/sequestration capacity.     

Mutations on the N-Terminal Edge of the DELSEED Loop in either the α or β Subunit of the Mitochondrial F1-ATPase Enhance ATP Hydrolysis in the Absence of the Central γ Rotor

F₁-ATPase is a rotary molecular machine with a subunit stoichiometry of α₃β₃γ₁δ₁ε₁. It has a robust ATP-hydrolyzing activity due to effective cooperativity between the three catalytic sites. It is believed that the central γ rotor dictates the sequential conformational changes to the catalytic sites in the α₃β₃ core to achieve cooperativity. However, recent studies of the thermophilic Bacillus PS3 F₁-ATPase have suggested that the α₃β₃ core can intrinsically undergo unidirectional cooperative catalysis (T. Uchihashi et al., Science 333:755-758, 2011). The mechanism of this γ-independent ATP-hydrolyzing mode is unclear. Here, a unique genetic screen allowed us to identify specific mutations in the α and β subunits that stimulate ATP hydrolysis by the mitochondrial F₁-ATPase in the absence of γ. We found that the F446I mutation in the α subunit and G419D mutation in the β subunit suppress cell death by the loss of mitochondrial DNA (ρ^o) in a Kluyveromyces lactis mutant lacking γ. In organello ATPase assays showed that the mutant but not the wild-type γ-less F₁ complexes retained 21.7 to 44.6% of the native F₁-ATPase activity. The γ-less F₁ subcomplex was assembled but was structurally and functionally labile in vitro. Phe446 in the α subunit and Gly419 in the β subunit are located on the N-terminal edge of the DELSEED loops in both subunits. Mutations in these two sites likely enhance the transmission of catalytically required conformational changes to an adjacent α or β subunit, thereby allowing robust ATP hydrolysis and cell survival under ρ^o conditions. This work may help our understanding of the structural elements required for ATP hydrolysis by the α₃β₃ subcomplex.     

Increased Expression of the Large Conductance,Calcium-Activated K+ (BK) Channel in Adult-Onset Neuronal Ceroid Lipofuscinosis

Cysteine string protein (CSPα) is a presynaptic J protein co-chaperone that opposes neurodegeneration. Mutations in CSPα (i.e., Leu115 to Arg substitution or deletion (Δ) of Leu116) cause adult neuronal ceroid lipofuscinosis (ANCL), a dominantly inherited neurodegenerative disease. We have previously demonstrated that CSPα limits the expression of large conductance, calcium-activated K⁺ (BK) channels in neurons, which may impact synaptic excitability and neurotransmission. Here we show by western blot analysis that expression of the pore-forming BKα subunit is elevated ~2.5 fold in the post-mortem cortex of a 36-year-old patient with the Leu116∆ CSPα mutation. Moreover, we find that the increase in BKα subunit level is selective for ANCL and not a general feature of neurodegenerative conditions. While reduced levels of CSPα are found in some postmortem cortex specimens from Alzheimer’s disease patients, we find no concomitant increase in BKα subunit expression in Alzheimer’s specimens. Both CSPα monomer and oligomer expression are reduced in synaptosomes prepared from ANCL cortex compared with control. In a cultured neuronal cell model, CSPα oligomers are short lived. The results of this study indicate that the Leu116∆ mutation leads to elevated BKα subunit levels in human cortex and extend our initial work in rodent models demonstrating the modulation of BKα subunit levels by the same CSPα mutation. While the precise sequence of pathogenic events still remains to be elucidated, our findings suggest that dysregulation of BK channels may contribute to neurodegeneration in ANCL.     

Variable DNA splicing sites of a mitochondrial intron: relationship to the senescence process in Podospora

The unavoidable phenomenon of senescence in Podospora was previously shown to be correlated with the presence of a senescence-specific DNA originating from amplification of some regions of the mitochondrial chromosome. The most frequently amplified region (α) corresponds to the first intron of the gene coding for subunit one of cytochrome oxidase. Eleven long-lived mitochondrial mutants were isolated. Here we report sequencing experiments that show that three of them are deleted for most of intron α and for a few base pairs belonging to the upstream adjacent exon. We also report an analysis of the residual mitochondrial DNA associated with amplification of senescence-specific DNA α which allows us to identify, in senescent cultures, mitochondrial chromosomes lacking sequence α. These results taken together suggest that excision of intron α from the mitochondrial DNA occurs systematically during the aging process in Podospora. They furthermore provide the first example of inaccurate intron excision at the DNA level.     

The mitochondrial DNA polymerase as a target of oxidative damage

The mitochondrial respiratory chain is a source of reactive oxygen species (ROS) that are responsible for oxidative modification of biomolecules, including proteins. Due to its association with mitochondrial DNA, DNA polymerase γ (pol γ) is in an environment to be oxidized by hydrogen peroxide and hydroxyl radicals that may be generated in the presence of iron ions associated with DNA. We tested whether human pol γ was a possible target of ROS with H₂O₂ and investigated the effect on the polymerase activities and DNA binding efficiency. A 1 h treatment with 250 µM H₂O₂ significantly inhibited DNA polymerase activity of the p140 subunit and lowered its DNA binding efficiency. Addition of p55 to the p140 catalytic subunit prior to H₂O₂ treatment offered protection from oxidative inactivation. Oxidatively modified amino acid residues in pol γ  resulting from H₂O₂ treatment were observed in vitro as well as in vivo, in SV40-transfected human fibroblasts. Pol γ was detected as one of the major oxidized mitochondrial matrix proteins, with a detectable decline in polymerase activity. These results suggest pol γ as a target of oxidative damage, which may result in a reduction in mitochondrial DNA replication and repair capacities.     

The Inhibitory Mechanism of the ζ Subunit of the F1FO-ATPase Nanomotor of Paracoccus denitrificans and Related α-Proteobacteria

The ζ subunit is a novel inhibitor of the F₁F_O-ATPase of Paracoccus denitrificans and related α-proteobacteria. It is different from the bacterial (ϵ) and mitochondrial (IF₁) inhibitors. The N terminus of ζ blocks rotation of the γ subunit of the F₁-ATPase of P. denitrificans (Zarco-Zavala, M., Morales-Ríos, E., Mendoza-Hernández, G., Ramírez-Silva, L., Pérez-Hernández, G., and García-Trejo, J. J. (2014) FASEB J. 24, 599–608) by a hitherto unknown quaternary structure that was first modeled here by structural homology and protein docking. The F₁-ATPase and F₁-ζ models of P. denitrificans were supported by cross-linking, limited proteolysis, mass spectrometry, and functional data. The final models show that ζ enters into F₁-ATPase at the open catalytic α_E/β_E interface, and two partial γ rotations lock the N terminus of ζ in an “inhibition-general core region,” blocking further γ rotation, while the ζ globular domain anchors it to the closed α_DP/β_DP interface. Heterologous inhibition of the F₁-ATPase of P. denitrificans by the mitochondrial IF₁ supported both the modeled ζ binding site at the α_DP/β_DP/γ interface and the endosymbiotic α-proteobacterial origin of mitochondria. In summary, the ζ subunit blocks the intrinsic rotation of the nanomotor by inserting its N-terminal inhibitory domain at the same rotor/stator interface where the mitochondrial IF₁ or the bacterial ϵ binds. The proposed pawl mechanism is coupled to the rotation of the central γ subunit working as a ratchet but with structural differences that make it a unique control mechanism of the nanomotor to favor the ATP synthase activity over the ATPase turnover in the α-proteobacteria.     

Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans

The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7. These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.     

A multifaceted cellular damage repair and prevention pathway promotes high‐level tolerance to β‐lactam antibiotics

Bactericidal antibiotics are powerful agents due to their ability to convert essential bacterial functions into lethal processes. However, many important bacterial pathogens are remarkably tolerant against bactericidal antibiotics due to inducible damage repair responses. The cell wall damage response two‐component system VxrAB of the gastrointestinal pathogen Vibrio cholerae promotes high‐level β‐lactam tolerance and controls a gene network encoding highly diverse functions, including negative control over multiple iron uptake systems. How this system contributes to tolerance is poorly understood. Here, we show that β‐lactam antibiotics cause an increase in intracellular free iron levels and collateral oxidative damage, which is exacerbated in the ∆vxrAB mutant. Mutating major iron uptake systems dramatically increases ∆vxrAB tolerance to β‐lactams. We propose that VxrAB reduces antibiotic‐induced toxic iron and concomitant metabolic perturbations by downregulating iron uptake transporters and show that iron sequestration enhances tolerance against β‐lactam therapy in a mouse model of cholera infection. Our results suggest that a microorganism''s ability to counteract diverse antibiotic‐induced stresses promotes high‐level antibiotic tolerance and highlights the complex secondary responses elicited by antibiotics.     

Reductive Evolution of the Mitochondrial Processing Peptidases of the Unicellular Parasites Trichomonas vaginalis and Giardia intestinalis

Mitochondrial processing peptidases are heterodimeric enzymes (α/βMPP) that play an essential role in mitochondrial biogenesis by recognizing and cleaving the targeting presequences of nuclear-encoded mitochondrial proteins. The two subunits are paralogues that probably evolved by duplication of a gene for a monomeric metallopeptidase from the endosymbiotic ancestor of mitochondria. Here, we characterize the MPP-like proteins from two important human parasites that contain highly reduced versions of mitochondria, the mitosomes of Giardia intestinalis and the hydrogenosomes of Trichomonas vaginalis. Our biochemical characterization of recombinant proteins showed that, contrary to a recent report, the Trichomonas processing peptidase functions efficiently as an α/β heterodimer. By contrast, and so far uniquely among eukaryotes, the Giardia processing peptidase functions as a monomer comprising a single βMPP-like catalytic subunit. The structure and surface charge distribution of the Giardia processing peptidase predicted from a 3-D protein model appear to have co-evolved with the properties of Giardia mitosomal targeting sequences, which, unlike classic mitochondrial targeting signals, are typically short and impoverished in positively charged residues. The majority of hydrogenosomal presequences resemble those of mitosomes, but longer, positively charged mitochondrial-type presequences were also identified, consistent with the retention of the Trichomonas αMPP-like subunit. Our computational and experimental/functional analyses reveal that the divergent processing peptidases of Giardia mitosomes and Trichomonas hydrogenosomes evolved from the same ancestral heterodimeric α/βMPP metallopeptidase as did the classic mitochondrial enzyme. The unique monomeric structure of the Giardia enzyme, and the co-evolving properties of the Giardia enzyme and substrate, provide a compelling example of the power of reductive evolution to shape parasite biology.     

Connexin 43 and metabolic effect of fatty acids in stressed endothelial cells

Changes in the inner mitochondrial membrane potential (∆ψ) may lead either to apoptosis or to protective autophagy. Connexin 43 (Cx43), a gap junction protein, is suggested to affect mitochondrial membrane permeability. The aim of our study was to analyze Cx43 gene expression, Cx43 protein localization and mitochondrial function in the human endothelial cells stressed by dietary-free fatty acids (FFA) and TNFα. Human endothelial cells (HUVECs) were incubated with (10–30 uM) palmitic (PA), oleic (OA), eicosapentaenoic (EPA) or arachidonic (AA) acids for 24 h. TNFα (5 ng/ml) was added at the last 4 h of incubation. The Cx43 gene expression was analyzed by the quantitative real-time PCR. The Cx43 protein concentrations in whole cells and in the isolated mitochondria were measured. Changes in ∆ψ and Cx43 localization were analyzed by flow cytometry or fluorescence microscopy. Generated ATP was measured by a luminescence assay. TNFα, PA and OA significantly decreased ∆ψ, while AA (P = 0.047) and EPA (P = 0.004) increased ∆ψ value. Preincubation with EPA or AA partially prevented the TNFα-induced decrease of ∆ψ. Incubation with AA resulted in up-regulation of the Cx43 gene expression. AA or PA significantly increased Cx43 protein content; however, presence of TNFα in general aggravated the negative effect of FFA. Only EPA was found to increase ATP generation in HUVECs. The fatty acid-specific induction of changes in Cx43 expression and protein concentration as well as the normalization of ∆ψ and increase of ATP generation seem to be the separate, independent mechanisms of FFA-mediated modulatory effect in the human endothelial cells pathology.     

A Highlights from MBoC Selection: The novel component Kgd4 recruits the E3 subunit to the mitochondrial α-ketoglutarate dehydrogenase

The mitochondrial citric acid cycle is a central hub of cellular metabolism, providing intermediates for biosynthetic pathways and channeling electrons to the respiratory chain complexes. In this study, we elucidated the composition and organization of the multienzyme complex α-ketoglutarate dehydrogenase (α-KGDH). In addition to the three classical E1-E3 subunits, we identified a novel component, Kgd4 (Ymr31/MRPS36), which was previously assigned to be a subunit of the mitochondrial ribosome. Biochemical analyses demonstrate that this protein plays an evolutionarily conserved role in the organization of mitochondrial α-KGDH complexes of fungi and animals. By binding to both the E1-E2 core and the E3 subunit, Kgd4 acts as a molecular adaptor that is necessary to a form a stable α-KGDH enzyme complex. Our work thus reveals a novel subunit of a key citric acid–cycle enzyme and shows how this large complex is organized.     

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis.     

The β Subunit of the Heterotrimeric G Protein Triggers the Kluyveromyces lactis Pheromone Response Pathway in the Absence of the γ Subunit

The Kluyveromyces lactis heterotrimeric G protein is a canonical Gαβγ complex; however, in contrast to Saccharomyces cerevisiae, where the Gγ subunit is essential for mating, disruption of the KlGγ gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Gγ, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and ΔGγ cells of K. lactis. In contrast to the moderate sterility shown by the single ΔKlGα, the double ΔKlGα ΔKlGγ mutant displayed full sterility. A partial sterile phenotype of the ΔKlGγ mutant was obtained in conditions where the KlGβ subunit interacted defectively with the Gα subunit. The addition of a CCAAX motif to the C-end of KlGβ, partially suppressed the lack of both KlGα and KlGγ subunits. In cells lacking KlGγ, the KlGβ subunit cofractionated with KlGα in the plasma membrane, but in the ΔKlGα ΔKlGγ strain was located in the cytosol. When the KlGβ-KlGα interaction was affected in the ΔKlGγ mutant, most KlGβ fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gβ subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Gγ subunit.