Novel mechanism coupling cyclic AMP-protein kinase A signaling and golgi trafficking via Gyp1 phosphorylation in polarized growth

Abstract

The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1(4E)) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1(4A)) abolished it. Gyp1(4E) exhibited enhanced association with the actin motor Myo2, while Gyp1(4A) showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1(R292K)) showed strong polarization similar to that seen with Gyp1(4E), indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.

FIG 1

Gyp1 functions in C. albicans hyphal growth. (A) gyp1Δ/Δ and gyp1^R292K strains have defects in hyphal development. Yeast cells of BWP17 (WT), HZX201 (gyp1Δ/Δ; for details on strain genotypes, please see Table S2 in the supplemental material), HZX250 (gyp5Δ/Δ), HZX251 (gyp8Δ/Δ), HZX202 (gyp1Δ/Δ:GYP1), and HZX203 (gyp1Δ/Δ:gyp1R292K) were grown overnight in YPD medium at 30°C, adjusted to an OD₆₀₀ of 0.1, and spotted onto Spider medium plates or Lee's medium plates. The plates were incubated for 3 days or 6 days, respectively. (B) The gyp1Δ/Δ mutant shows markedly slower hyphal extension in liquid media. As shown in the left panel, cells of BWP17 (WT) and HZX201 (gyp1Δ/Δ) were induced for hyphal development in GMM–20% fetal bovine serum (FBS) at 37°C for 2 h before imaging. The right panel shows the distributions of hyphae with different ratios of hyphal length to the diameter of the mother cell body at 2 h of hyphal induction (n = 120).

FIG 2

Gyp1 localizes to the Golgi compartment and is required for Golgi polarization in C. albicans. (A) Localization of Gyp1-GFP in WT (HZX202) and bni1Δ/Δ (HZX225) yeast and hyphal cells. Yeast cells were grown in GMM to the log phase at 30°C. Hyphal cells were grown in GMM–20% FBS at 37°C for 2 h. Size bar = 5 μm. Images of differential interference contrast (DIC) and GFP fluorescence microscopy results are shown. (B) Localization of Vrg4 in short and long hyphae in WT (HZX210) and gyp1Δ/Δ (HZX211) backgrounds. Hyphal cells were grown in GMM–20% FBS at 37°C for 2 to 3 h. Although gyp1Δ/Δ cells are defective in hyphal growth, a small percentage of long hyphae can be found. Size bar = 5 μm in all images.

FIG 3

C. albicans Gyp1 is a PKA substrate. (A) Gyp1 is phosphorylated by PKA upon hyphal induction. Log-phase yeast cells expressing Gyp1-GFP (HZX202) were subjected to hyphal induction by switching from GMM at 30°C to prewarmed GMM–20% serum at 37°C. Aliquots were harvested at the indicated times after hyphal induction for preparation of cell lysates. PKA phosphorylation of Gyp1 was detected by Western blot analysis using the anti-PKA substrate antibody, and Gyp1 was probed with anti-GFP antibody. (B) Gyp1 can be phosphorylated by bovine PKA in vitro. Gyp1-GFP immunoprecipitated from yeast cells (HZX202) was subjected to in vitro kinase assay in the presence (+) or absence (−) of bovine PKA and then subjected to Western blot analysis using anti-PKA substrate antibody and anti-GFP antibody. (C and D) Gyp1 PKA phosphorylation levels are decreased in cyr1Δ/Δ and tpk2Δ/Δ cells upon hyphal induction. WT (HZX202), cyr1Δ/Δ (HZX232), tpk1Δ/Δ (HZX226), and tpk2Δ/Δ (HZX227) cells expressing Gyp1-GFP were processed and PKA phosphorylation of Gyp1 was analyzed as described for panel A.

FIG 4

PKA phosphorylation of Gyp1 controls its polarized localization. (A) The diagram depicts the location of and amino acid sequences around four PKA-phosphorylated serines (Sp) identified by mass spectrometry in this study. (B) Gyp1^4A is less phosphorylated than WT Gyp1 by bovine PKA in vitro. A kinase assay was performed, and PKA phosphorylation of Gyp1 was analyzed as described for Fig. 2C. PKA phosphorylation of Gyp1 was quantified by dividing the density of the PKA substrate band (PKA phos) by that of the Gyp1 band (lower panel). The experiment was done three times, and the difference between PKA phosphorylation of Gyp1 and that of Gyp1^4A was statistically significant (*, P < 0.05 [t test]). Error bars represent standard errors of the means (SEM). (C) Hyphal development of GYP1 phosphorylation mutants (phosphomutants) on solid medium. A hyphal development assay was performed on Spider medium plates as described for Fig. 1A. The strains used were HZX202 (gyp1Δ/Δ GYP1), HZX204 (gyp1Δ/Δ gyp1^4A), and HZX205 (gyp1Δ/Δ gyp1^4E). (D) Cellular localization of Gyp1^4A-GFP (HZX204) and Gyp1^4E-GFP (HZX205) in yeast and hyphal cells.

FIG 5

PKA phosphorylation and GAP activity inactivation of Gyp1 enhance its association with Myo2. (A) Log-phase yeast cells coexpressing Myo2-hemagglutinin (Myo2-HA) with Gyp1 (HZX232), Gyp1-GFP (HZX228), Gyp1^4A-GFP (HZX230), Gyp1^4E-GFP (HZX231), or Gyp1^R292K-GFP (HZX229) were subjected to immunoprecipitation (IP) with anti-HA antibody to pull down Myo1, and the precipitation products were analyzed by Western blotting using anti-GFP and anti-HA antibody. The band intensity of the GFP-tagged Gyp1 protein was normalized to the band intensity of Myo2-HA in the same sample (lower panel). The experiment was done three times, and the differences between WT Gyp1 and mutant Gyp1 proteins were statistically significant (*, P < 0.05; **, P < 0.01 [t test]). Error bars represent SEM. (B and C) Localization of Gyp1^R292K-GFP (HZX203) and Gyp1^4A,R292K-GFP (HZX233) in yeast and hyphal cells.

FIG 6

Recruitment of Sec7 to late Golgi compartments requires PKA phosphorylation of Gyp1. Actively growing yeast and hyphal cells of HZX215 (GYP1 SEC7-GFP) (A), HZX216 (gyp1Δ/Δ SEC7-GFP) (B), HZX218 (gyp1^4A SEC7-GFP) (C), and HZX219 (gyp1^4E SEC7-GFP) (D) were examined for Sec7-GFP localization by fluorescence microscopy. Yeast cells were grown in GMM at 30°C, and hyphae were induced in GMM–20% FBS at 37°C for 2 h.

FIG 7

Gyp1 function is required for virulence of C. albicans. (A) Survival curves of mice inoculated with 1 × 10⁶ yeast cells with the indicated genotype. Eight mice were used for each C. albicans strain (SC5314, HZX201, HZX202, HZX204, HZX205, and HZX203). (B) Kidney fungal burden of the infected mice. Two mice for each C. albicans strain were sacrificed 2 days after injection to determine the log₁₀CFU/kidney (*, P < 0.01 [t test]). (C) Histological examination of the kidneys of infected mice. The kidneys of mice infected with C. albicans with the indicated genotypes were removed 2 days after infection. Kidney sections were stained using the periodic acid-Schiff staining method, which stains C. albicans cells dark magenta. The square in the 1× image marks that region shown in the 40× image.