Vacuolar Trafficking Protein VPS38 Is Dispensable for Autophagy

Abstract

Phosphatidylinositol 3-P (PI3P) is a signaling molecule that controls a variety of processes in endosomal, autophagic, and vacuolar/lysosomal trafficking in yeasts and mammals. Vacuolar protein sorting 34 (Vps34) is a conserved PI3K present in multiple complexes with specific functions and regulation. In yeast, the PI3K complex II consists of Vps34p, Vps15p, Vps30p/ Atg6p, and Vps38p, and is essential for vacuolar protein sorting. Here, we describe the Arabidopsis (Arabidopsis thaliana) homolog of yeast Vps38p and human UV radiation resistance-associated gene protein. Arabidopsis VPS38 interacts with VPS30/ATG6 both in yeast and in planta. Although the level of PI3P in Arabidopsis vps38 mutants is similar to that in wild type, vps38 cells contain enlarged multivesicular endosomes and fewer organelles enriched in PI3P than the wild type. The vps38 mutants are defective in the trafficking of vacuolar cargo and its receptor VACUOLAR SORTING RECEPTOR2;1. The mutants also exhibit abnormal cytoplasmic distributions of endocytic cargo, such as auxin efflux carriers PINFORMED1 (PIN1) and PIN2. Constitutive autophagy is normal in the mutants but starvation-induced autophagy was slightly inhibited. We conclude that Arabidopsis VPS38 is dispensable for autophagy but essential for efficient targeting of biosynthetic and endocytic cargo to the vacuole.

The Vacuolar Protein Sorting-38 Subunit of the Arabidopsis Phosphatidylinositol-3-Kinase Complex Plays Critical Roles in Autophagy, Endosome Sorting, and Gravitropism

“…Strikingly, homozygous Arabidopsis vps38 mutants are viable but display a range of defects consistent with aberrant endosomal sorting, vacuolar trafficking, and autophagy (this report; Lee et al, 2018 ), which are consistent with studies with yeast and mammalian cells that demonstrated the importance of PtdIn-3P to these processes ( Kihara et al, 2001 ; Itakura et al, 2008 ). In our study, the defects were confirmed by a pair of vps38 alleles, along with their rescue by a VPS38-FLAG transgene.…”

“…To study the function(s) of Arabidopsis VPS38 genetically, we searched the available germplasm collections for T-DNA mutants impacting the locus. Two mutant lines were available from the Arabidopsis Biological Resource Center (ABRC) which were confirmed by DNA sequence analysis; one contains a T-DNA insertion within the 2nd exon after nucleotide 507 downstream of the start codon (SAIL_552_F02), which was designated here as vps38-1 , and a second contains a T-DNA insertion within the 5th exon after nucleotide 1,886 downstream of the translation start site (SALK_094540C), which was designated as vps38-3 based on the allele nomenclature of Lee et al (2018) ( Figure 3A ). Homozygous individuals were identified in the selfed F2 generation by PCR genotyping, indicating that plants missing VPS38, unlike mutants impacting other subunits of the class-III PtdIn-3 kinase complex ( Fujiki et al, 2007 ; Qin et al, 2007 ; Harrison-Lowe and Olsen, 2008 ; Lee et al, 2008b ; Xu et al, 2011 ; Wang et al, 2012 ), produce viable pollen that will germinate.…”

“…Given that a functional ATG14 remains in vps38 plants, we presume that the class-III PtdIn-3 kinase complex I assembled with ATG14 provides the necessary PtdIn-3P to maintain plant cell viability in the absence of VPS38. In support, Lee et al (2018) analyzed the profile of PtdIns in the vps38-1 and vsp38-2 backgrounds and found no changes in PtdIn-3P abundance and only slight increases in PtdIn-4P and bis-phosphorylated PtdIn-4,5P 2 forms. Clearly, studies of triple mutants missing VPS38, ATG14a, and ATG14b will be necessary to address the essential nature of this subunit in the class-III complex.…”

“…We, and a recent study by Lee et al (2018) , overcame this hurdle by examining mutants eliminating the accessory subunit VPS38, which exchanges with ATG14 to distinguish the complex-I and complex-II isoforms ( Kihara et al, 2001 ; Itakura et al, 2008 ). Although amino acid sequence similarities between VPS38 from Arabidopsis and other plants and their yeast and mammalian orthologs are low, both the predicted presence of the signature BARA2 and coiled-coil domains in the plant sequences ( Rostislavleva et al, 2015 ), and our demonstration by several interaction assays that Arabidopsis VPS38 interacts with the VPS34, VPS15, and ATG6 subunits of the core class-III complex confirmed its identity.…”

“…Problems in the plasma membrane to endosome cycling of the PIN-FORMED (PIN) auxin efflux carriers necessary for tropic curvature were observed, thus connecting VPS38 to the endocytosis and vesicular trafficking pathways that control these carriers ( Kleine-Vehn et al, 2008 ; Di Sansebastiano et al, 2014 ; Adamowski and Friml, 2015 ). Together with a companion paper by Lee et al (2018) , genetic analysis of the non-essential VPS38 subunit reveals that the class-III PtdIn-3 kinase complex has pleiotropic roles in membrane identity and intracellular trafficking in plants.…”