TheArabidopsisMultistress Regulator TSPO Is a Heme Binding Membrane Protein and a Potential Scavenger of Porphyrins via an Autophagy-Dependent Degradation Mechanism  

Abstract

TSPO, a stress-induced, posttranslationally regulated, early secretory pathway-localized plant cell membrane protein, belongs to the TspO/MBR family of regulatory proteins, which can bind porphyrins. This work finds that boosting tetrapyrrole biosynthesis enhanced TSPO degradation in Arabidopsis thaliana and that TSPO could bind heme in vitro and in vivo. This binding required the His residue at position 91 (H91), but not that at position 115 (H115). The H91A and double H91A/H115A substitutions stabilized TSPO and rendered the protein insensitive to heme-regulated degradation, suggesting that heme binding regulates At-TSPO degradation. TSPO degradation was inhibited in the autophagy-defective atg5 mutant and was sensitive to inhibitors of type III phosphoinositide 3-kinases, which regulate autophagy in eukaryotic cells. Mutation of the two Tyr residues in a putative ubiquitin-like ATG8 interacting motif of At-TSPO did not affect heme binding in vitro but stabilized the protein in vivo, suggesting that downregulation of At-TSPO requires an active autophagy pathway, in addition to heme. Abscisic acid-dependent TSPO induction was accompanied by an increase in unbound heme levels, and downregulation of TSPO coincided with the return to steady state levels of unbound heme, suggesting that a physiological consequence of active TSPO downregulation may be heme scavenging. In addition, overexpression of TSPO attenuated aminolevulinic acid-induced porphyria in plant cells. Taken together, these data support a role for TSPO in porphyrin binding and scavenging during stress in plants.

The ATG1/ATG13 Protein Kinase Complex Is Both a Regulator and a Target of Autophagic Recycling in Arabidopsis

“…While it was first thought to be nonspecific, recent studies have identified routes for selective autophagy (Xie and Klionsky, 2007;Noda et al, 2008;Behrends et al, 2010). Such selectivity likely provides a critical housekeeping function by removing damaged chloroplasts, mitochondria (mitophagy), and ribosomes (ribophagy), scavenging free porphyrins, clearing unwanted peroxisomes (and possibly glyoxysomes) as their available substrate pools change (pexophagy), degrading ubiquitylated aggregates too large for the 26S proteasome, and possibly even sequestering pathogens that invade the cytosol (Ishida et al, 2008;Wada et al, 2009;Hillwig et al, 2011;Johansen and Lamark, 2011;Vanhee et al, 2011;Youle and Narendra, 2011).…”

Ultrastructure of autophagy in plant cells

“…The cargo receptor (AtNBR1) thus seems to be a selective autophagy substrate, just as the molecules it binds to. 36 The data of Vanhee et al 35 and Svenning et al, 36 however, were not accompanied by an ultrastructural correlate.…”

“…It thus seems that both the carrier (TSPO) and the carried product become degraded. 35 Another example was described by Svenning et al…”

“…For example, Vanhee et al 35 reported that in Arabidopsis the controlled degradation of tryptophan-rich sensory protein (TSPO) was inhibited in ATG5 mutant plants. TSPO is a secretory pathway cell membrane protein.…”

A BAR-Domain Protein SH3P2, Which Binds to Phosphatidylinositol 3-Phosphate and ATG8, Regulates Autophagosome Formation in Arabidopsis

“…Since SH3P2 has a membrane-remodeling domain, it is likely that an interaction between SH3P2 and ATG8 promotes membrane deformation for developing autophagosome. Recently, selective autophagy has been identified in plants, and the ATG8 family interacting motif has emerged as the molecular basis for ATG8 to recognize cargo(s) or other proteins in regulating autophagy (Suttangkakul et al, 2011;Svenning et al, 2011;Vanhee et al, 2011;Derrien et al, 2012;Floyd et al, 2012;Honig et al, 2012). Since SH3P2 signals were detected inside the vacuole after autophagy induction (Figures 1Bc and 1Bd), such an SH3P2-ATG8 interaction may target SH3P2 for degradation to regulate the turnover of autophagy.…”