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. 2006 May;173(1):49-61.
doi: 10.1534/genetics.106.055699. Epub 2006 Feb 19.

Schizosaccharomyces pombe Git1 is a C2-domain protein required for glucose activation of adenylate cyclase

Richard S Kao  1 , Eric Morreale, Lili Wang, F Douglas Ivey, Charles S Hoffman
Affiliations

Schizosaccharomyces pombe Git1 is a C2-domain protein required for glucose activation of adenylate cyclase

Richard S Kao et al. Genetics. 2006 May.

Abstract

Schizosaccharomyces pombe senses environmental glucose through a cAMP-signaling pathway, activating cAMP-dependent protein kinase A (PKA). This requires nine git (glucose insensitive transcription) genes that encode adenylate cyclase, the PKA catalytic subunit, and seven "upstream" proteins required for glucose-triggered adenylate cyclase activation, including three heterotrimeric G-protein subunits and its associated receptor. We describe here the cloning and characterization of the git1+ gene. Git1 is distantly related to a small group of uncharacterized fungal proteins, including a second S. pombe protein that is not functionally redundant with Git1, as well as to members of the UNC-13/Munc13 protein family. Mutations in git1+ demonstrate functional roles for the two most highly conserved regions of the protein, the C2 domain and the MHD2 Munc homology domain. Cells lacking Git1 are viable, but display phenotypes associated with cAMP-signaling defects, even in strains expressing a mutationally activated G alpha-subunit, which activates adenylate cyclase. These cells possess reduced basal cAMP levels and fail to mount a cAMP response to glucose. In addition, Git1 and adenylate cyclase physically interact and partially colocalize in the cell. Thus, Git1 is a critical component of the S. pombe glucose/cAMP pathway.

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Figures

Figure 1.
Figure 1.
Plasmid pAF1 (his3+) insertion mutation is in the git1+ complementation group. Diploid strains were constructed using intragenically complementing ade6-M210 and ade6-M216 mutant alleles to form Ade+ diploids. Diploid strains were spotted onto 5-FOA medium and grown for 3 days at 30°. 5-FOAR growth reflects the wild-type ability to glucose repress transcription of an fbp1-ura4+ reporter gene.
Figure 2.
Figure 2.
Complementation of a git1-1 mutation by plasmid-expressed git1+. Strains FWP72 (git1+) and CHP568 (git1-1) were transformed to Leu+ with pNMT41 (empty vector), pRSK3 (git1+), pRSK4 (git1-V5his6), or pRSK8 (git1ΔC2). Three independent transformants of each host strain and plasmid combination indicated in the figure were spotted to PM–leu and then replica plated after 2 days to either PM–leu or 5-FOA media. Plates were photographed after 3 days at 30°.
Figure 3.
Figure 3.
Multicopy suppression of git2− mutations by plasmid-expressed git1+. Host strains CHP7 (git2-7), CHP61 (git2-61), and CHP216 (git2-216) were transformed to Leu+ with pNMT41 (empty vector) or pRSK3 (git1+). Four independent transformants of each host strain and plasmid combination indicated in the figure were plated onto EMM–leu and then replica plated after 1 day to either EMM–leu or 5-FOA media. 5-FOA plates were photographed after 5 days at 30°. All transformants grew equally well on EMM–leu medium (not shown). β-Galactosidase activity was determined as described in materials and methods. The values given represent specific activity ± standard deviation from three independent transformants.
Figure 4.
Figure 4.
Alignment of Git1 with SPAC11E3.02c and related proteins from Ustilago maydis and Yarrowia lipolytica. (A) Alignment of S. pombe proteins Git1 (NP_596600) and SPAC11E3.02c (NP_594926) with related proteins from U. maydis (XP_400071) and Y. lipolytica (XP_501312) using the ClustalW sequence alignment program (Thompson et al. 1994) and displayed using BOXSHADE. Identities are shown against a solid background and similarities are shown against a shaded background. A shaded box above the sequences denotes the predicted C2 domain of Git1. The alanine-to-aspartic-acid missense mutation in git1-45 is shown above residue 799 of the Git1 sequence. Asterisks above the Git1 sequence at residues 995 and 1022 denote the last correctly translated residues expressed from alleles git1-1 and git1-79, respectively. (B) E-values associated with alignments from unfiltered BLASTP searches involving the proteins shown in A.
Figure 5.
Figure 5.
Git1 protein levels are regulated by glucose conditions. Western blot analysis of protein extracts from FWP72 (Git+) transformants carrying plasmid pRSK4 (Git1+-V5) or plasmid pRSK9 (Git1ΔC2-V5). Cells were grown under glucose-repressing (R) or derepressing (D; glucose-starved) conditions. Immunoblots were carried out using either α-V5 or α-Cdc2 (as a loading control) antibody as described in materials and methods.
Figure 6.
Figure 6.
Spore germination delay in git1Δ by git1Δ cross. Thirteen tetrads from each cross were dissected on yeast extract agar plates and incubated at 30°. Photographs were taken on days 3, 4, 5, and 11. Crosses (from left to right) are of strains FWP95 (git1+) × FWP87 (git1+), FWP95 × CHP954 (git1Δ), RKP2 (git1Δ) × FWP87, and RKP2 × CHP954.
Figure 7.
Figure 7.
git1Δ cells display defects associated with the loss of cAMP signaling. (A) Homothallic (h90) strains CHP795 (git1+) and CHP896 (git1Δ) were grown to exponential phase in PM liquid medium (at 37° to inhibit conjugation) and then diluted to 106 cells/ml in PM liquid medium in the presence or absence of 5 mm cAMP. These cells were incubated for 24 hr at 30° without shaking and photographed. (B) Strains 972 (git1+) and CHP973 (git1Δ) were grown to exponential phase in YEL. Cells displaying septa were measured and photographed.
Figure 8.
Figure 8.
Git1 and Git2 (adenylate cyclase) physically interact. (A) Immunoblot of protein extracts from α-V5 immunoprecipitation. Protein extracts were prepared from strains expressing tagged (Git2-myc and Git1-V5) or untagged proteins (+) as indicated. Crude extracts, along with fractions that bound or failed to bind α-V5 antibodies, were probed with α-myc to detect Git2-myc and with α-V5 to detect Git1-V5. (B) Immunoblot of protein extracts from α-myc immunoprecipitation. Protein extracts were prepared from strains expressing tagged or untagged proteins as indicated. Crude extracts, along with fractions that bound or failed to bind α-myc antibodies were probed with α-V5 to detect Git1-V5 and with α-myc to detect Git2-myc.
Figure 9.
Figure 9.
Partial colocalization of Git1 and Git2. (A) DIC and fluorescent images of cells expressing tagged and untagged forms of Git1 and Git2 (adenylate cyclase) as indicated. Fluorescent images were taken with the same exposure time for each sample. (B) Merged fluorescent image from A of cells expressing Git1-V5 and Git2-myc.
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