Localization of RNS2 ribonuclease to the vacuole is required for its role in cellular homeostasis
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Localization of the RNase RNS2 to the vacuole via a C-terminal targeting signal is essential for its function in rRNA degradation and homeostasis.
RNase T2 ribonucleases are highly conserved enzymes present in the genomes of nearly all eukaryotes and many microorganisms. Their constitutive expression in different tissues and cell types of many organisms suggests a housekeeping role in RNA homeostasis. The Arabidopsis thaliana class II RNase T2, RNS2, is encoded by a single gene and functions in rRNA degradation. Loss of RNS2 results in RNA accumulation and constitutive activation of autophagy, possibly as a compensatory mechanism. While the majority of RNase T2 enzymes is secreted, RNS2 is located within the vacuole and in the endoplasmic reticulum (ER), possibly within ER bodies. As RNS2 has a neutral pH optimum, and the endomembrane organelles are connected by vesicle transport, the site within the endomembrane system at which RNS2 functions is unclear. Here we demonstrate that localization to the vacuole is essential for the physiological function of RNS2. A mutant allele of RNS2, rns2-1, results in production of an active RNS2 RNase but with a mutation that removes a putative C-terminal vacuolar targeting signal. The mutant protein is, therefore, secreted from the cell. This results in a constitutive autophagy phenotype similar to that observed in rns2 null mutants. These findings illustrate that the intracellular retention of RNS2 and localization within the vacuole are critical for its cellular function.
KeywordsArabidopsis Autophagy Ribosomal RNA RNA degradation Vacuolar targeting
This work was supported by Grant No. MCB-1051818 from the United States National Science Foundation to GCM and DCB and Grant No. DE-SC0014038 from the United States Department of Energy to DCB. We thank Danielle Ebany for isolation of an rns2-1 homozygote and Junmarie Soto-Burgos for assistance with confocal microscopy.
- Ahmed SU, Rojo E, Kovaleva V, Venkataraman S, Dombrowski JE, Matsuoka K, Raikhel NV (2000) The plant vacuolar sorting receptor AtELP is involved in transport of NH2-terminal propeptide-containing vacuolar proteins in Arabidopsis thaliana. J Cell Biol 149:1335–1344. doi: 10.1083/jcb.149.7.1335 CrossRefPubMedPubMedCentralGoogle Scholar
- Fuji K, Shirakawa M, Shimono Y, Kunieda T, Fukao Y, Koumoto Y, Takahashi H, Hara-Nishimura I, Shimada T (2016) The adaptor complex AP-4 regulates vacuolar protein sorting at the trans-Golgi network by interacting with VACUOLAR SORTING RECEPTOR1. Plant Physiol 170(1):211–219. doi: 10.1104/pp.15.00869 CrossRefPubMedGoogle Scholar
- Haud N, Kara F, Diekmann S, Henneke M, Willer JR, Hillwig MS, Gregg RG, Macintosh GC, Gartner J, Alia A, Hurlstone AF (2011) rnaset2 mutant zebrafish model familial cystic leukoencephalopathy and reveal a role for RNase T2 in degrading ribosomal RNA. Proc Natl Acad Sci USA 108(3):1099–1103. doi: 10.1073/pnas.1009811107 CrossRefPubMedPubMedCentralGoogle Scholar
- Henneke M, Diekmann S, Ohlenbusch A, Kaiser J, Engelbrecht V, Kohlschutter A, Kratzner R, Madruga-Garrido M, Mayer M, Opitz L, Rodriguez D, Ruschendorf F, Schumacher J, Thiele H, Thoms S, Steinfeld R, Nurnberg P, Gartner J (2009) RNASET2-deficient cystic leukoencephalopathy resembles congenital cytomegalovirus brain infection. Nat Genet 41(7):773–775. doi: 10.1038/ng.398 CrossRefPubMedGoogle Scholar
- Kang H, Kim SY, Song K, Sohn EJ, Lee Y, Lee DW, Hara-Nishimura I, Hwang I (2012) Trafficking of vacuolar proteins: the crucial role of Arabidopsis vacuolar protein sorting 29 in recycling vacuolar sorting receptor. Plant Cell 24(12):5058–5073. doi: 10.1105/tpc.112.103481 CrossRefPubMedPubMedCentralGoogle Scholar
- Lehmann K, Hause B, Altmann D, Kock M (2001) Tomato ribonuclease LX with the functional endoplasmic reticulum retention motif HDEF is expressed during programmed cell death processes, including xylem differentiation, germination, and senescence. Plant Physiol 127(2):436–449CrossRefPubMedPubMedCentralGoogle Scholar
- MacIntosh GC, Bariola PA, Newbigin E, Green PJ (2001) Characterization of Rny1, the Saccharomyces cerevisiae member of the T2 RNase family of RNases: unexpected functions for ancient enzymes? Proc Natl Acad Sci USA 98(3):1018–1023. doi: 10.1073/pnas.98.3.1018 CrossRefPubMedPubMedCentralGoogle Scholar
- Sheen J (2002) A transient expression assay using Arabidopsis mesophyll protoplasts. Available online at http://molbio.mgh.harvard.edu/sheenweb/protocols_reg.html
- Yamamoto H, Kakuta S, Watanabe TM, Kitamura A, Sekito T, Kondo-Kakuta C, Ichikawa R, Kinjo M, Ohsumi Y (2012) Atg9 vesicles are an important membrane source during early steps of autophagosome formation. J Cell Biol 198(2):219–233. doi: 10.1083/jcb.201202061 CrossRefPubMedPubMedCentralGoogle Scholar