Elsevier

Gene

Volume 391, Issues 1–2, 15 April 2007, Pages 233-241
Gene

Characterization of a novel alternatively spliced human transcript encoding an N-terminally truncated Vps24 protein that suppresses the effects of Bax in an ESCRT independent manner in yeast

https://doi.org/10.1016/j.gene.2006.12.039Get rights and content

Abstract

Elucidating novel anti-apoptotic regulatory pathways is central to further understanding the molecular basis of several pathologies, including cancer. We have previously reported the identification of several mammalian cDNAs effective in preventing the lethal effects of heterologous expression of a pro-apoptotic BAX cDNA in yeast [Yang, Z., Khoury, C., Jean-Baptiste, G., Greenwood, M.T., 2006. Identification of mouse sphingomyelin synthase 1 (SMS1) as a suppressor of Bax mediated cell death in yeast. FEMS Yeast Res. 6, 751–762]. Here we report that one of the Bax suppressors encodes a novel 156 amino acid variant of the human Vps24 protein, Vps24β, that lacks the N-terminal lipid binding domain of the well characterized 222 residue Vps24 (Vps24α). We demonstrate that the VPS24β cDNA represents an expressed transcript that is likely produced by alternative splicing of the human VPS24 gene. Vps24α, but not Vps24β, prevented the temperature and salt sensitive growth defects observed in a yeast mutant lacking a functional VPS24 gene. In contrast, Vps24β, but not Vps24α, suppressed the inhibitory effects of Bax on yeast growth. Vps24β protein also suppressed the effects of Bax in mutants lacking other VPS genes suggesting that a functional ESCRT pathway, of which the yeast Vps24p is an essential component, is not required for Vps24β function. Taken together, we demonstrate that the human VPS24 gene gives rise to two functionally distinct proteins, one of which is involved in the ESCRT pathway and another novel protein that serves an anti-apoptotic role.

Introduction

Anti-apoptosis can be operationally defined as the collective set of mechanisms that cells use to evade or delay the apoptotic program. Many regulators of anti-apoptosis have been discovered during the past decade, allowing the elucidation of specific pathways that promote cell survival. Some such proteins, such as the anti-apoptotic Bcl-2 family members, the IAPs, and Akt, serve to directly antagonize the pro-apoptotic cascade (Kumar et al., 2002, Reed et al., 2004). Other proteins that have been shown to have anti-apoptotic function by buffering stimulus-induced cellular alterations, and include heat shock proteins (hsp; i.e. Hsp72, Hsp90) or antioxidant enzymes (i.e. Superoxide dismutase) (Kumar et al., 2002, Takayama et al., 2003). Further, a number of other proteins with poorly understood or uncharacterized functions such as the GTPase GIMAP8 and matrix metalloproteinase 15 (MMP-15) also confer resistance to apoptotic stimuli when over-expressed in cultured cells (Abraham et al., 2005, Krucken et al., 2005). Ostensibly, attaining an exhaustive list of anti-apoptotic regulators precludes a full understanding of the process. Identifying new anti-apoptotic proteins therefore remains an important endeavor.

Of late, a number of novel anti-apoptotic sequences have been identified by laboratories using functional screens in the yeast Saccharomyces cerevisiae (Greenhalf et al., 1999, Levine et al., 2001, Pan et al., 2001, Xu and Reed, 1998, Yang et al., 2006). Yeast cells undergo a programmed cell death response to a variety of different stressful stimuli that shares many characteristic features with metazoan apoptosis, including chromatin condensation, phosphatidylserine externalization and ROS accumulation (Ludovico et al., 2005, Madeo et al., 2004). Additionally, both heterologous expressions of the pro-apoptotic Bax and acetic acid were shown to elicit the release of cytochrome c from the mitochondria, an established mammalian apoptogenic event (Ludovico et al., 2002, Manon et al., 1997). Indeed, yeast orthologues of known regulators of metazoan apoptosis have been discovered, including YCA1, AIF1 and NMA111 (Fahrenkrog et al., 2004, Madeo et al., 2002, Wissing et al., 2004) and appear to be functionally conserved. These findings, among others, suggest the use of yeast as a powerful and genetically tractable model for the study of apoptosis.

We have previously reported the isolation of 62 different mammalian cDNAs that were capable of blocking the effects of Bax in yeast (Yang et al., 2006). In that study we identified and characterized the mouse sphingomyelin synthase 1 (SMS1) gene as a sequence capable of preventing cell death in response to multiple stresses. Cells that are stressed often undergo apoptosis due to increases in the intracellular levels of second messengers such as ROS and the pro-apoptotic sphingolipid ceramide. The anti-apoptotic property of SMS1 is likely related to its ability to use ceramide as a precursor for the synthesis of sphingomyelin (Yang and Greenwood, 2005). Thus the identification of novel anti-apoptotic sequences that have a well-known function can serve to implicate specific cellular processes as well as being useful as tools in the study of apoptotic pathways. Here we provide a detailed characterization of another Bax-suppressive clone encoding a novel alternatively spliced variant of vesicle sorting protein 24 (Vps24). Vps24 has been previously identified as an essential component of the ESCRT complexes that are involved in sorting protein cargo to the lysosome or vacuole (Babst, 2005). The alternatively spliced Vps24 variant identified as a Bax suppressor is predicted to lack the N-terminal 66 residues that are known to contain the lipid binding domain of the previously characterized 222 residue Vps24 protein (Whitley et al., 2003). Our analysis of both Vps24 proteins in yeast suggests that the 222 residue human Vps24 protein can only function as an ESCRT protein, while the truncated Vps24 can only function as an anti-apoptotic protein. Thus the alternative splicing of the human Vps24 gives rise to two functionally distinct proteins.

Section snippets

Yeast strains and plasmids

Strain BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was used as the wild type strain. All deletion mutants were isogenic to BY4741 and were obtained from EUROSCARF (http://web.uni-frankfurt.de/fb15/mikro/euroscarf/index.html). The Bax suppressors were previously isolated by screening a human heart expression cDNA library in yeast cells expressing Bax under the control of the GAL1 promoter (Yang et al., 2006). The heart cDNA library and consequently all the Bax suppressors used in this study are

Vacuolar protein sorting 24β (VPS24β) is an alternatively spliced variant of the VPS24 gene

Vps24 is a component of endosomal sorting complex required for transport (ESCRT) involved in multivesicular body formation (MVB) that serves to transport protein cargo to the lysosome (Babst, 2005). Although the lysosome and vesicle trafficking have been implicated in the process of apoptotic programmed cell death, ESCRT has not (Babst, 2005, Gonzalez-Polo et al., 2005, Levine et al., 2001). We identified a VPS24 cDNA in our previously described screen for Bax suppressors (Yang et al., 2006).

Conclusion

This work describes a novel protein product of the human VPS24 gene, VPS24β, that suppresses the effects of multiple apoptotic stimuli in yeast. We provide evidence to suggest that VPS24β is generated by the alternative splicing of exon 2, ultimately resulting in the production of an N-terminally truncated protein with respect to the previously characterized Vps24α. It was demonstrated that the two isoforms are functionally distinct. The function of Vps24β was also shown to be independent of

Acknowledgements

This work was supported by the Canadian Institute of Health Research (CIHR), the Natural Sciences and Engineering Research Council of Canada (NSERC) and the Heart and Stroke Foundation (HSF) of Quebec. M.T.G. is the recipient of a Charles O. Monat Scholarship (McGill University), Z.Y. is in part supported by a Scholarship from the McGill University Hospital Center (MUHC), and C.M.K. is the recipient of a post-graduate Scholarship from the MUHC. We thank Dr. C. Mandato and X.-Y. Li for technical

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