The Arabidopsis exocyst subcomplex subunits involved in a golgi-independent transport into the vacuole possess consensus autophagy-associated atg8 interacting motifs

Abstract

The exocyst complex is a multi-subunits evolutionary conserved complex, which was originally shown to be primarily associated with vesicular transport to the plasma membrane. A recent report (Kulich et al., 2013 Traffic; In Press) revealed that AtEXO70B1, one of the multiple subunits of the exocyst complex of Arabidopsis thaliana plants, is co-transported with the autophagy-associated Atg8f protein to the vacuole. This pathway does not involve the Golgi apparatus. The co-localization of AtEXO70B1 and Atg8f suggests either that both of these proteins are co-transported together to the vacuole or, alternatively, that Atg8 binds to a putative Atg8 interacting motif (AIM) located within the AtEXO70B1 polypeptide, apparently forming a tethering complex for an autophagic complex that is transported to the vacuole. In the present addendum, by tooling a bioinformatics approach, we show that AtEXO70B1 as well as the additional 20 paralogs of Arabidopsis EXO70 exocyst subunits each possess one or more AIMs whose consensus sequence implies their high fidelity binding to Atg8. This indicates that the autophagy machinery is strongly involved in the assembly, transport, and apparently also the function of AtEXO70B1 as well as the exocyst sub complex.

Keywords: :

• Arabidopsis
• Autophagy
• Atg8
• vacuole
• Exocyst

Much information regarding the patterns and mechanisms of trafficking of plant proteins from the endoplasmic reticulum (ER) to the vacuole has been accumulated in the literature. A significant amount of the data has been derived from studies using seed storage proteins as model systems. Classically, plant proteins that reside in the vacuole are transported to this organelle from the ER via the Golgi apparatus. Yet, based on multiple studies using various microscopic tools, it was proposed that the storage proteins of seeds from several plant species are also transported directly from the ER to protein storage vacuoles (PSVs) by a route that may bypass the Golgi and may be reminiscent of macroautophagy (hereafter referred to as autophagy).^1-9 A similar Golgi-bypass transport of seed storage from the ER to the vacuole has also been reported in germinating Vinga Mango seedlings.^10,11 Furthermore, our laboratory has recently reported an additional support for a direct autophagy-associated ER-to-vacuole transport, using Arabidopsis seedlings exposed to carbon starvation.^12,13 This report identified a novel AIM-containing plant protein, termed ATI1, which has been proven to bind the central autophagy-associated protein Atg8 using various approaches. In a plant grown under optimal, non-stress conditions, ATI1 is partially associated with the ER. In response to carbon starvation ATI1 becomes associated with a newly identified spherical compartment that scans the ER network and is subsequently transported to the vacuole. The transported compartment contains ATI1 bound to Atg8 on its surface.

In a recent report, Kulich et al.,¹⁴ showed that a subunit of the exocyst complex of Arabidopsis plants (EXO70B1) is co-transported with Atg8f to the vacuole by an intra-cellular route that may apparently be associated with the autophagy machinery. This was based on the observation that AtEXO70B1 and Atg8f were co-localized along their transport to the vacuoles. The exact goal of the transport of EXO70B1 to the vacuole has not been addressed in this report. One possible goal is that EXO70B1 was trans located to the vacuole as a cargo for its own degradation in this organelle. Alternatively, EXO70B1 could serve as a tethering component of the machinery that trans locates cargo components to the vacuole. Nevertheless, whether the AtEXO70B1 directly interacts with Atg8f along this transport route, an interaction that would indicate the direct involvement of autophagy in this process has not been addressed in this report. To address this issue, we have used a bioinformatics approach to elucidate whether AtEXO70B1 possesses a consensus Atg8 binding motif (AIM) that would apparently mediate its binding to Atg8f and hence its transport to the vacuole using the autophagy machinery. We also analyzed further whether any of other 20 Arabidopsis exocyst subunits possess AIMs. The amino acid sequences of all of these exocyst subunits were retrieving from the protein sequence database of PUBMED (http://www.ncbi.nlm.nih.gov/pubmed/).

Consensus AIMs¹⁵ include a core of 4 amino acids in which the first amino acid is either F, W, or Y and the fourth amino acid is either L, I, or V. The 2 amino acids in between the F/W/Y and L/I/V can be any amino acids with preference to acidic amino acids. In addition, the presence of acidic amino in the 3 positions upstream the F/W/Y, and/or the 2 positions between F/W/Y and L/I/V and/or the 3 positions downstream the L/I/V stimulate the potential of the protein containing such AIM to bind to Atg8. The closer the up stream acidic amino acids to F/W/Y and the closer the down stream acidic amino acids to L/I/V, the stronger is the fidelity of binding of Atg8 to this AIM. For simplicity, the AIM can also be schematically described as “X_-3X_-2X_-1[WYF]X₊₁X₊₂[VIL]X₊₃X₊₄X₊₅“ in which the acidic amino acids are preferred within the X positions (positions marked in sub-script numbers). Serine (S) and threonine (T) present in the vicinity of AIMs or even within them provide negative charges and hence they may also replace the acidic amino acids needed for the function of the AIM.¹⁵ However, this depends on the number of S and T amino acids as their localization within the AIM. Taking into account the degenerate nature of AIMs, we first defined a number of degenerated AIM motifs without integrating S or T into the AIM (Table S1). We then considered each of these motifs as potentially functional AIMs that bind Atg8 at a reasonably high fidelity. Next, we adapted the stand-alone version of the PatMatch software¹⁶ (http://www.ncbi.nlm.nih.gov/pubmed/15980466) in order to identify within a given protein the AIMs that meet any of the consensus motifs described in Table S1. PatMach is an effective web-based pattern-matching program allowing searching for short nucleotide and protein sequences, such as short consensus motifs, and therefore is suitable for identifying AIMs. This program is available for download at The Arabidopsis Information Resource (TAIR) at ftp://ftp.Arabidopsis.org/home/tair/Software/Patmatch/. Finally, we also manually searched nearby the core [WFY][XXL[IV] sequences of the AIM motifs for the presence of potential nearby stretches of S or T residues to define additional potentially functional AIMs. Scanning the amino acid sequence of AtEXO70B1 (624 amino acids) by this bioinformatics approach recognized 5 independent AIMs that meet the standards described in Table S1 (Fig. 1; AIMs are indicated by bolded underlined letters). Thus, we concluded that the co-localization of AtEXO70B1 with AtAtg8f, as observed in the confocal microscope,¹⁴ is potentially due to the interaction of these 2 proteins via the binding of Atg8f to any of the AIMs present in AtEXO70B1.

Figure 1. Elucidation of Atg8-binding motifs in the Arabidopsis exocyst subunit exo70 family protein H7. The amino acid sequence of this protein was retrieved from the protein database of PUBMED. The Atg8-binding motifs (AIMs) were elucidated by a bioinformatics approach, as described in the text. This approach identified 2 consensuses AIM motif, which are marked in bold letters.

In their report,¹⁴ Kulich and associates also showed that AtEXO70B1 appears to possess completely different functions from those of AtEXO70A1. It was thus interesting to test whether AtEXO70A1 possess AIMs and, if yes, whether the AIMs are located in similar or distinct regions within the amino acid sequences of these 2 proteins. As shown in Figure 1 despite their different functions, each of these 2 proteins possessed 1 AIM having similar amino acid sequence and being localized in a similar region within the amino acid sequence of the proteins. Yet, as shown further in Figure 1 each of these 2 proteins also possessed additional AIMs, which are distinct in their numbers and locations within the amino acid sequences of the 2 proteins. This implies that the apparent different functions of these 2 exocyst subunits, as indicated by Kulich et al., are associated with apparent distinct association of these proteins with the autophagy process.

Next, we selected the 20 additional independent Arabidopsis exocyst protein subunits existing in the protein database of PUBMED (http://www.ncbi.nlm.nih.gov/pubmed/) and analyzed their amino acid sequences for AIMs by PatMatch (ftp://ftp.Arabidopsis.org/home/tair/Software/Patmatch/).¹⁶ We also further proofed the AIMs manually and checked further for potential AIMs that may include S and T residues either within or in proximity to the core F/W/YXXLI/V AIM. The names and amino acid sequences of these 20 Arabidopsis exocyst proteins are provided in a FASTA format in Figure S1. Notably, all of these Arabidopsis exocyst proteins possessed AIMs meeting the consensus sequences described in Table S1. Furthermore, the numbers of the AIMs per individual protein ranged between 1 to 5 motifs, and in some of the proteins the AIMs also included S and T residues (Figure S1). Our demonstration that AtEXO70B1 possesses 5 AIMs (4 of which are high-confidence AIMs based on the consensus AIM motif shown in Figure 1), when taken together with the co-localization of AtEXO70B1 with Atg8f, supports the notion that the autophagy machinery participates in the transport of the protein complex containing AtEXO70B1 to the vacuole. Our further results (Fig. S1) also imply that not only AtEXO70B1, but also multiple other Arabidopsis exocyst subunits possess consensus AIMs, further indicating a major regulatory role for Atg8 and likely also other components of the plant autophagy machinery in the transport of the exocyst subunits to the vacuole.

Although this article deals exocyst subunits, the existence of AIMs is not specific to these subunits. A number of other Arabidopsis proteins, possessing diverse functions, were shown to contain AIMs, implying that selective autophagy is intensely involved in plant growth and response to different cues, particularly to environmental stresses. Three examples of such functionally diverse Atg8-binding proteins are ATI,¹³ TSPO,¹⁷ and the autophagic cargo receptor NBR1.¹⁸ ATI1 is an Arabidopsis Atg8-binding protein defining a newly identified stress-induced compartment that originates on the endoplasmic reticulum. In response to carbon starvation, the ATI1 compartment is transported to the vacuole, apparently transferring yet unknown cargo from the ER to the vacuole. Overexpression or suppression of ATI1 gene expression stimulates or suppresses seed germination on medium containing the germination-suppressing hormone ABA. TSPO is a multi-stress regulator heme binding protein potentially serving as a scavenger of porphyrins. NBR1 is a selective autophagic cargo receptor diverting proteins that were originally destined for degradation by the proteasome to the autophagy machinery. Such a process may optimize cargo degradation, using these 2 compartments.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgements

Our research was supported by a grant from the J and R center for Scientific Research at the Weizmann Institute of Science and the Israeli Ministry of Agriculture. GG is an incumbent of the Bronfman Chair at the Weizmann Institute of Science.

Supplementary Material

Supplementary material may be found here: http://www.landesbioscience.com/journals/psb/article/26732/