Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family.

Yeasts and fungi generate Ca2+ signals in response to environmental stresses through Ca2+ channels essentially composed of Cch1 and Mid1. Cch1 is homologous to the pore-forming α1 subunit of animal voltage-gated Ca2+ channels (VGCCs) and sodium leak channels nonselective (NALCNs), whereas Mid1 is a membrane-associated protein similar to the regulatory α2/δ subunit of VGCCs and the regulatory subunit of NALCNs. Although the physiological roles of Cch1/Mid1 channels are known, their molecular regulation remains elusive, including subunit interactions regulating channel functionality. Herein, we identify amino acid residues involved in interactions between the pore-forming Cch1 subunit and the essential regulatory Mid1 subunit of Saccharomyces cerevisiaeIn vitro mutagenesis followed by functional assays and co-immunoprecipitation experiments reveal that three residues present in a specific extracellular loop in the repeat III region of Cch1 are required for interaction with Mid1, and that one essential Mid1 residue is required for interaction with Cch1. Importantly, these residues are necessary for Ca2+ channel activity and are highly conserved in fungal and animal counterparts. We discuss that this unique subunit interaction-based regulatory mechanism for Cch1 differs from that of VGCCs/NALCNs.

A bipolar functionality of Q/N-rich proteins: Lsm4 amyloid causes clearance of yeast prions.

Prions are epigenetic modifiers that cause partially loss-of-function phenotypes of the proteins in Saccharomyces cerevisiae. The molecular chaperone network that supports prion propagation in the cell has seen a great progress in the last decade. However, the cellular machinery to activate or deactivate the prion states remains an enigma, largely due to insufficient knowledge of prion-regulating factors. Here, we report that overexpression of a [PSI(+) ]-inducible Q/N-rich protein, Lsm4, eliminates the three major prions [PSI(+) ], [URE3], and [RNQ(+) ]. Subcloning analysis revealed that the Q/N-rich region of Lsm4 is responsible for the prion loss. Lsm4 formed an amyloid in vivo, which seemed to play a crucial role in the prion elimination. Fluorescence correlation spectroscopy analysis revealed that in the course of the Lsm4-driven [PSI(+) ] elimination, the [PSI(+) ] aggregates undergo a size increase, which ultimately results in the formation of conspicuous foci in otherwise [psi(-) ]-like mother cells. We also found that the antiprion activity is a general property of [PSI(+) ]-inducible factors. These data provoked a novel "unified" model that explains both prion induction and elimination by a single scheme.

Clearance of yeast prions by misfolded multi-transmembrane proteins.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the stress response to protect cells against toxicity by the unfolded protein response (UPR), heat shock response (HSR), and ER-associated degradation pathways. Here, we found that over-production of C-terminally truncated multi-transmembrane (MTM) mutant proteins triggers HSR, but not UPR, and clearance of yeast prions [PSI(+)] and [URE3]. One of the mutant MTM proteins, Dip5ΔC-v82, produces a disabled amino-acid permease. Fluorescence microscopy analysis revealed abnormal accumulation of Dip5ΔC-v82 in the ER. Importantly, the mutant defective in the GET pathway, which functions for ER membrane insertion of tail-anchored proteins, failed to translocate Dip5ΔC-v82 to the ER and disabled Dip5ΔC-v82-mediated prion clearance. These findings suggest that the GET pathway plays a pivotal role in quality assurance of MTM proteins, and entraps misfolded MTM proteins into ER compartments, leading to loss-of-prion through a yet undefined mechanism.

A bipolar personality of yeast prion proteins.

Prions are infectious, self-propagating protein conformations. [PSI+], [RNQ+] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1, and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI+] prion in [RNQ+] or [URE3] strain, but not in non-prion strain. However, [RNQ+] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1Δ100, and Lsm4, hamper the maintenance of [PSI+]. These findings point to a bipolar activity of [RNQ+], [URE3], Rnq1Δ100, and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI+] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed.

[PSI(+)] aggregate enlargement in rnq1 nonprion domain mutants, leading to a loss of prion in yeast.

[PIN(+)] is the prion form of the Rnq1 protein of unknown function in Saccharomyces cerevisiae. A glutamine/asparagine (Q/N)-rich C-terminal domain is necessary for the propagation of [PIN(+)], whereas the N-terminal region is non-Q/N-rich and considered the nonprion domain. Here, we isolated numerous single-amino-acid mutations in Rnq1, phenotypically similar to Rnq1Δ100, which inhibit [PSI(+)] propagation in the [PIN(+)] state, but not in the [pin(-)] state, when overproduced. The dynamics of the prion aggregates was analyzed by semi-denaturing detergent-agarose gel electrophoresis and fluorescence correlation spectroscopy. The results indicated that [PSI(+)] aggregates were enlarged in mother cells and, instead, not apparently transmitted into daughter cells. Under these conditions, the activity of Hsp104, a known prion disaggregase, was not affected when monitored for the thermotolerance of the rnq1 mutants. These [PSI(+)]-inhibitory rnq1 mutations did not affect [PIN(+)] propagation itself when over-expressed from a strong promoter, but instead destabilized [PIN(+)] when expressed from the weak authentic RNQ1 promoter. The majority of these mutated residues are mapped to the surface, and on one side, of contiguous α-helices of the nonprion domain of Rnq1, suggesting its involvement in interactions with a prion or a factor necessary for prion development.

Halogen bonds as orthogonal molecular interactions to hydrogen bonds.

Halogen bonds (X-bonds) are shown to be geometrically perpendicular to and energetically independent of hydrogen bonds (H-bonds) that share a common carbonyl oxygen acceptor. This orthogonal relationship is accommodated by the in-plane and out-of-plane electronegative potentials of the oxygen, which are differentially populated by H- and X-bonds. Furthermore, the local conformation of a peptide helps to define the geometry of the H-bond and thus the oxygen surface that is accessible for X-bonding. These electrostatic and steric forces conspire to impose a strong preference for the orthogonal geometry of X- and H-bonds. Thus, the optimum geometry of an X-bond can be predicted from the pattern of H-bonds in a folded protein, enabling X-bonds to be introduced to improve ligand affinities without disrupting these structurally important interactions. This concept of orthogonal molecular interactions can be exploited for the rational design of halogenated ligands as inhibitors and drugs, and in biomolecular engineering.