PMC

The binA gene and its predicted protein. (A) The binA gene (VFA1038) is located downstream from and oriented in the same direction as the syp gene locus. Individual genes are indicated by block arrows, and the four known or putative promoters within the syp locus are indicated by line arrows. (B) The BinA protein (630 amino acids [aa]) is predicted to have three domains, GAF (∼Q20 to L151), GGDEF (∼H205 to A338), and EAL (∼L374 to D611), as indicated. Only the EAL domain is well conserved.

Cleavage sites of HRA1 RNA with yeast RNase P. (A) 5′-end-labeled HRA1 RNA was a substrate for yeast RNase P cleavage in vitro. An alkali ladder and partial RNase T1 digestion of HRA1 RNA, as described in Materials and Methods, were used for comparison with the cleavage products. Yeast HRA1 RNA cleaved by yeast RNase P holoenzyme, alkali ladder of HRA1 RNA, and partial RNase T1 digestion of HRA1 RNA were individually precipitated with ethanol with glycogen as carrier, resuspended with 8 M urea dye, and loaded on to 6% polyacrylamide/8 M urea sequencing gel. After separating, the gel was dried and exposed to BioMax film (Kodak). The major and minor cleavage sites were marked with arrows. (B) The major cleavage site was located at the 5′ side of G404 (underlined), and the minor cleavage is a mixture of several cleavages at the 5′ termini of three adjacent nucleotides from A338 to G340 (bold and underlined), which were located in the antisense region that overlapped with the DRS2 gene (bold). (C) Scheme of cleavage of HRA1 RNA with major and minor cleavage sites (indicated by arrows). The secondary structure of HRA1 RNA was estimated using the MFOLD program (Zuker et al. 1999). Only individual nucleotides near the cleavage sites are shown.

Pfh1p is present in three distinct isoforms. (A) Schematic of Pfh1p ORF and predicted isoforms drawn to scale. MTS, predicted mitochondrial targeting signal; MIP, predicted mitochondrial import processing site. Methionine codons upstream of helicase domain are denoted by amino acid positions 1, 21, 170, 265, and 320. Helicase motifs are indicated by roman numerals; the invariant lysine K338 in helicase motif I (denoted by ^) is mutated to A338 in the catalytically dead (cd) allele. The predicted translation products from methionine codons M1 and M21 in WT and mutant strains are indicated. (B) Whole-cell anti-Pfh1p Western blot in untagged and GFP-tagged pfh1 strains. unt, pfh1⁺; WT, pfh1⁺-GFP; m1, pfh1-m1-GFP; m21, pfh1-m21-GFP. Here and in subsequent figures, S, I, and F denote top, middle, and bottom Pfh1p bands. S′, I′, and F′ denote top, middle, and bottom Pfh1p-GFP bands. The faint band below (F) is seen in a subset of gels and is likely a degradation product. +CPT, camptothecin. LC, loading control blot probed with anti-Rdp1 polyclonal serum. Molecular mass markers here and in subsequent figures are given in kilodaltons.

Signal in the endodomain of gI encompassing T³³⁸ is necessary for targeting expressed protein to the TGN. The nomenclature and corresponding sequences of the gI mutant constructs used to transfect Cos-7 cells are depicted in Fig. 2. The arrows indicate fluorescence in the TGN region of transfected cells. (A) Expressed gI_wt is concentrated in the TGN and plasma membrane. (B) Expressed gI_δ1 (lacking the five C-terminal amino acids of gI_wt) is still targeted to the TGN and plasma membrane. (C and D) Expressed gI_δ2 (lacking the nine C-terminal amino acids of gI_wt) and gI_δ3 (lacking the 12 C-terminal amino acids of gI_wt) are still targeted to the TGN and plasma membrane. (E) Expressed gI_δ2KK (E³⁴¹ to K³⁴¹ and E³⁴² to K³⁴²) is targeted to the TGN and plasma membrane. This mutation destroys a consensus casein kinase II site. (F) Expressed gI_δR→E (R³⁴⁰ to E³⁴⁰; also lacking the 12 C-terminal amino acids of gI_wt) is targeted to the TGN and plasma membrane. R³⁴⁰ is thus not a component of a TGN targeting signal. (G) Expressed gI_δ3t→A (T³³⁸ to A³³⁸; also lacking the 12 C-terminal amino acids of gI_wt) is not targeted to the TGN or plasma membrane. The pattern of immunoreactivity suggests that the expressed gI_δ3t→A may be retained in the RER. The same results are obtained when T³³⁸ is mutated to S³³⁸ or D³³⁸ (not illustrated). T³³⁸ is thus required for TGN targeting. (H to K) Expressed gI_δ4, gI_δ5, gI_δ6, and gI_δ7, respectively (which lack, respectively, the 17, 19, 36, and 59 [entire endodomain] C-terminal amino acids of gI_wt and thus the critical T³³⁸), are not targeted to the TGN or plasma membrane. Note that gI_δ5 contains all of the amino acids N-terminal to T³³⁸. (L) Expressed gI_δcd+tm is concentrated in the TGN. Note that gI_δcd+tm differs from gI_δ7 (K), which lacks the endodomain of gI_wt, in that the transmembrane domain of gI_wt is also deleted in gI_δcd+tm. Bars, 10 μm.