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Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

Vauchelles R, Stalder D, Botton T, Arkowitz RA, Bassilana M - PLoS ONE (2010)

Bottom Line: Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus.Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1.Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Developmental Biology and Cancer, Centre National de la Recherche Scientifique UMR 6543, Université de Nice, Faculté des Sciences-Parc Valrose, Nice, France.

ABSTRACT
The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

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Rac1 polybasic carboxyl-terminal region is required for membrane localization and function.(A) Rac1 PBR is required for plasma membrane localization. DIC and fluorescence images of indicated strains rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511), and rac1Δ/rac1Δ PADH1GFPrac1[C233S] (PY415), respectively, are shown. Bar, 5 µm. (B) Rac1 PBR is required for nuclear accumulation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511) incubated 75 min without agitation are shown. Bar, 5 µm. (C) In the absence of prenylation, the Rac1 carboxyl-terminal region targets GFP to the nucleus. Fluorescence images of indicated strains (PY357 and PY438, respectively) are shown. Bar, 5 µm. (D) Rac1 PBR is required for function. Cells from rac1Δ/rac1Δ PRAC1RAC1 (PY275), rac1Δ/rac1Δ PRAC1rac1-5Q (PY534), and rac1Δ/rac1Δ PRAC1rac1[C233S] (PY406) were embedded in YEPS and images of colonies were taken, as described in Figure 3C. Similar results were observed in 3 independent experiments. Bar, 1 mm.
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pone-0015400-g004: Rac1 polybasic carboxyl-terminal region is required for membrane localization and function.(A) Rac1 PBR is required for plasma membrane localization. DIC and fluorescence images of indicated strains rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511), and rac1Δ/rac1Δ PADH1GFPrac1[C233S] (PY415), respectively, are shown. Bar, 5 µm. (B) Rac1 PBR is required for nuclear accumulation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511) incubated 75 min without agitation are shown. Bar, 5 µm. (C) In the absence of prenylation, the Rac1 carboxyl-terminal region targets GFP to the nucleus. Fluorescence images of indicated strains (PY357 and PY438, respectively) are shown. Bar, 5 µm. (D) Rac1 PBR is required for function. Cells from rac1Δ/rac1Δ PRAC1RAC1 (PY275), rac1Δ/rac1Δ PRAC1rac1-5Q (PY534), and rac1Δ/rac1Δ PRAC1rac1[C233S] (PY406) were embedded in YEPS and images of colonies were taken, as described in Figure 3C. Similar results were observed in 3 independent experiments. Bar, 1 mm.

Mentions: In human cells, the Rac1 carboxyl-terminal region has been shown to be important both for localization and function. In particular, a nuclear localization sequence (NLS) in the HsRac1 carboxyl-terminal polybasic region (PBR) was identified and shown to be required for nuclear accumulation [12], [13]. Based on the consensus NLS motif (K-K/R-X-K/R), we identified two potential NLS's in C. albicans Rac1 carboxyl-terminal PBR: K225KRK228 and K230RAK233. To investigate the role of these sequences in Rac1 sub-cellular localization, we initially generated strains expressing GFP-Rac1-5Q (Q225QQKIQQ231, replacing K225KRKIKR231). We also generated a strain expressing GFP-Rac1[C233S], which cannot be geranylgeranylated. Figure 4A shows that GFP-Rac1-5Q was not observed at the plasma membrane and instead was present in the cytoplasm as well as associated with internal membranes. This Rac1-5Q mutant did not accumulate in the nucleus in the absence of cell agitation (Figure 4B). In contrast, GFP-Rac1[C233S] accumulated predominantly in the nucleus (Figure 4A) in the same condition. In addition, while GFP fused to the 14 carboxyl-terminal residues of Rac1 (GFP-CTRac1) did not accumulate in the nucleus in the absence of cell agitation (Figure 3A), mutation of Cys at position 233 to Ser (GFP-CTRac1[C233S]) resulted in nuclear accumulation of this fusion (Figure 4C). These results indicate that the NLS motifs present in the PBR are necessary and sufficient for targeting Rac1 to the nucleus in C. albicans, and suggest that the function of these NLS sequences is blocked or overridden by geranylgeranyl modification, as has been proposed for mammalian cells [10]. As expected if Rac1 functions primarily at the plasma membrane, rac1-5Q and rac1[C233S] were unable to restore embedded filamentous growth in a rac1 deletion mutant (Figure 4D). Together, these results indicate that the carboxyl-terminal polybasic region of C. albicans Rac1, similar to that of HsRac1, is necessary for its localization and function.


Rac1 dynamics in the human opportunistic fungal pathogen Candida albicans.

Vauchelles R, Stalder D, Botton T, Arkowitz RA, Bassilana M - PLoS ONE (2010)

Rac1 polybasic carboxyl-terminal region is required for membrane localization and function.(A) Rac1 PBR is required for plasma membrane localization. DIC and fluorescence images of indicated strains rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511), and rac1Δ/rac1Δ PADH1GFPrac1[C233S] (PY415), respectively, are shown. Bar, 5 µm. (B) Rac1 PBR is required for nuclear accumulation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511) incubated 75 min without agitation are shown. Bar, 5 µm. (C) In the absence of prenylation, the Rac1 carboxyl-terminal region targets GFP to the nucleus. Fluorescence images of indicated strains (PY357 and PY438, respectively) are shown. Bar, 5 µm. (D) Rac1 PBR is required for function. Cells from rac1Δ/rac1Δ PRAC1RAC1 (PY275), rac1Δ/rac1Δ PRAC1rac1-5Q (PY534), and rac1Δ/rac1Δ PRAC1rac1[C233S] (PY406) were embedded in YEPS and images of colonies were taken, as described in Figure 3C. Similar results were observed in 3 independent experiments. Bar, 1 mm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2965673&req=5

pone-0015400-g004: Rac1 polybasic carboxyl-terminal region is required for membrane localization and function.(A) Rac1 PBR is required for plasma membrane localization. DIC and fluorescence images of indicated strains rac1Δ/rac1Δ PADH1GFPRAC1 (PY205), rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511), and rac1Δ/rac1Δ PADH1GFPrac1[C233S] (PY415), respectively, are shown. Bar, 5 µm. (B) Rac1 PBR is required for nuclear accumulation. DIC and fluorescence images of rac1Δ/rac1Δ PADH1GFPrac1-5Q (PY511) incubated 75 min without agitation are shown. Bar, 5 µm. (C) In the absence of prenylation, the Rac1 carboxyl-terminal region targets GFP to the nucleus. Fluorescence images of indicated strains (PY357 and PY438, respectively) are shown. Bar, 5 µm. (D) Rac1 PBR is required for function. Cells from rac1Δ/rac1Δ PRAC1RAC1 (PY275), rac1Δ/rac1Δ PRAC1rac1-5Q (PY534), and rac1Δ/rac1Δ PRAC1rac1[C233S] (PY406) were embedded in YEPS and images of colonies were taken, as described in Figure 3C. Similar results were observed in 3 independent experiments. Bar, 1 mm.
Mentions: In human cells, the Rac1 carboxyl-terminal region has been shown to be important both for localization and function. In particular, a nuclear localization sequence (NLS) in the HsRac1 carboxyl-terminal polybasic region (PBR) was identified and shown to be required for nuclear accumulation [12], [13]. Based on the consensus NLS motif (K-K/R-X-K/R), we identified two potential NLS's in C. albicans Rac1 carboxyl-terminal PBR: K225KRK228 and K230RAK233. To investigate the role of these sequences in Rac1 sub-cellular localization, we initially generated strains expressing GFP-Rac1-5Q (Q225QQKIQQ231, replacing K225KRKIKR231). We also generated a strain expressing GFP-Rac1[C233S], which cannot be geranylgeranylated. Figure 4A shows that GFP-Rac1-5Q was not observed at the plasma membrane and instead was present in the cytoplasm as well as associated with internal membranes. This Rac1-5Q mutant did not accumulate in the nucleus in the absence of cell agitation (Figure 4B). In contrast, GFP-Rac1[C233S] accumulated predominantly in the nucleus (Figure 4A) in the same condition. In addition, while GFP fused to the 14 carboxyl-terminal residues of Rac1 (GFP-CTRac1) did not accumulate in the nucleus in the absence of cell agitation (Figure 3A), mutation of Cys at position 233 to Ser (GFP-CTRac1[C233S]) resulted in nuclear accumulation of this fusion (Figure 4C). These results indicate that the NLS motifs present in the PBR are necessary and sufficient for targeting Rac1 to the nucleus in C. albicans, and suggest that the function of these NLS sequences is blocked or overridden by geranylgeranyl modification, as has been proposed for mammalian cells [10]. As expected if Rac1 functions primarily at the plasma membrane, rac1-5Q and rac1[C233S] were unable to restore embedded filamentous growth in a rac1 deletion mutant (Figure 4D). Together, these results indicate that the carboxyl-terminal polybasic region of C. albicans Rac1, similar to that of HsRac1, is necessary for its localization and function.

Bottom Line: Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus.Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1.Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

View Article: PubMed Central - PubMed

Affiliation: Institute of Developmental Biology and Cancer, Centre National de la Recherche Scientifique UMR 6543, Université de Nice, Faculté des Sciences-Parc Valrose, Nice, France.

ABSTRACT
The small Rho G-protein Rac1 is highly conserved from fungi to humans, with approximately 65% overall sequence identity in Candida albicans. As observed with human Rac1, we show that C. albicans Rac1 can accumulate in the nucleus, and fluorescence recovery after photobleaching (FRAP) together with fluorescence loss in photobleaching (FLIP) studies indicate that this Rho G-protein undergoes nucleo-cytoplasmic shuttling. Analyses of different chimeras revealed that nuclear accumulation of C. albicans Rac1 requires the NLS-motifs at its carboxyl-terminus, which are blocked by prenylation of the adjacent cysteine residue. Furthermore, we show that C. albicans Rac1 dynamics, both at the plasma membrane and in the nucleus, are dependent on its activation state and in particular that the inactive form accumulates faster in the nucleus. Heterologous expression of human Rac1 in C. albicans also results in nuclear accumulation, yet accumulation is more rapid than that of C. albicans Rac1. Taken together our results indicate that Rac1 nuclear accumulation is an inherent property of this G-protein and suggest that the requirements for its nucleo-cytoplasmic shuttling are conserved from fungi to humans.

Show MeSH
Related in: MedlinePlus