The Retromer, Sorting Nexins and the Plant Endomembrane Protein Trafficking Nicole Heucken and Rumen Ivanov*

REVIEW SPECIAL ISSUE: PLANT CELL BIOLOGY The retromer, sorting nexins and the plant endomembrane protein trafficking Nicole Heucken and Rumen Ivanov*

ABSTRACT to distinct subdomains of the compartment. This shows that the Protein sorting in the endomembrane system is responsible for the many functions of the TGN might, to a certain extent, be spatially coordination of cellular functions. Plant intracellular trafficking has its separated (Bassham et al., 2000; Sanderfoot et al., 2001; Staehelin own unique features, which include specific regulatory aspects of and Kang, 2008). endosomal sorting and recycling of cargo proteins, mediated by the The late endosome is a structure with smaller internal (luminal) retromer complex. Recent work has led to significant progress in vesicles, giving it the name multivesicular body (MVB). From understanding the role of Arabidopsis retromer subunits in recycling the TGN, proteins performing functions in the tonoplast or in the vacuolar sorting receptors and plasma membrane proteins. As a vacuole, or those targeted for vacuolar degradation, pass through the consequence, members of the sorting nexin (SNX) protein family and MVB (Fig. 1). The MVB was shown to originate from the TGN their interaction partners have emerged as critical protein trafficking (Scheuring et al., 2011), supporting the idea that endomembrane regulators, in particular with regard to adaptation to environmental compartments represent continuous stages, rather than static structures change, such as temperature fluctuations and nutrient deficiency. In (Robinson and Neuhaus, 2016). However, it needs to be noted that other this Review, we discuss the known and proposed functions of the transport routes are known, such as secretion from the Golgi to the PM comparatively small Arabidopsis SNX protein family. We review the bypassing the TGN and direct ER-to-vacuole transport (Crowell et al., available information on the role of the three Bin-Amphiphysin-Rvs 2009; Viotti et al., 2013), whose prominence remains to be determined. (BAR)-domain-containing Arabidopsis thaliana (At)SNX proteins and Transport between compartments is bidirectional and proteins discuss their function in the context of their potential participation in may undergo retrograde transport towards a preceding trafficking the plant retromer complex. We also summarize the role of AtSNX1- stage (Fig. 1). A form of retrograde transport is the recycling of interacting proteins in different aspects of SNX-dependent protein vacuolar sorting receptors (VSRs) and PM proteins. In the first case, trafficking and comment on the potential function of three novel, as yet soluble cargo proteins, such as acid hydrolases, move towards the unexplored, Arabidopsis SNX proteins. vacuole owing to their interaction with VSRs already at the ER (Kunzl et al., 2016; Niemes et al., 2010a). The conditions in the KEY WORDS: Retromer, Sorting nexin, Protein sorting, Vacuolar TGN lumen promote the dissociation of the complex, and whereas sorting receptor, Transporter recycling, Environmental response the soluble cargos proceed with the flow towards the vacuole, the VSRs are transported backwards (recycled) towards the ER (Kunzl Introduction et al., 2016; Robinson and Neuhaus, 2016). Similarly, endocytosed The correct distribution of proteins in the endomembrane system is PM proteins – such as transporters and transmembrane receptors – critical for the maintenance of cellular functions and the survival of can be recycled back to the PM by being actively diverted from the the organism. In plants, trafficking towards the plasma membrane default vacuolar degradation pathway (Barberon et al., 2011; (PM) or the vacuole is a multistep process occurring through several Dhonukshe et al., 2007; Ivanov et al., 2014; Kasai et al., 2011; Luo intracellular compartments. Transmembrane or soluble luminal et al., 2015; Viotti et al., 2010). The wealth of often contradictory proteins are synthesized at the endoplasmic reticulum (ER) and data suggests that the sorting events that underlie these recycling transported towards the Golgi (Fig. 1). The cis-Golgi cisternae processes occur at the TGN and most probably involve the early accept material from the ER and gradually mature. Ultimately, they stages of MVB maturation (Robinson and Neuhaus, 2016). form a new tubular-vesicular structure, the trans-Golgi network The retromer is a key protein complex involved in cargo recycling (TGN), which contains the ER-derived proteins. In plants, the TGN and retrograde transport. Its components were identified in screens exists close to the trans-Golgi face, but also as a Golgi-independent for yeast (Saccharomyces cerevisiae) mutants defective in vacuolar compartment (Kang et al., 2011; Viotti et al., 2010). It is a major trafficking (Paravicini et al., 1992; Seaman et al., 1998). The hub where the two transport routes – one leading to the PM and the retromer consists of two subcomplexes, the core retromer and the other to the vacuole – are separated. The TGN fulfills the role of an sorting nexin (SNX) subcomplex (Fig. 2A). In this Review, we early endosome (Dettmer et al., 2006; Lam et al., 2007), and is also discuss the role of the retromer complex and SNXs in plant protein responsible for sorting and recycling material to or from the Golgi, sorting. By drawing comparison to the yeast and mammalian the PM and the lytic pathway (Gu et al., 2001; Kunzl et al., 2016; systems, we outline the common and specific functions of the plant Luo et al., 2015; Paez Valencia et al., 2016; Reguera et al., 2015). retromer. We further concentrate on the plant SNX protein family, Consistent with this, certain TGN-localized protein markers localize which consists of three previously known and three novel, as yet uncharacterized, proteins, and we discuss their localization, Institute of Botany, Heinrich-Heine University, Universitätsstrasse 1, 40225 regulation and functions. Düsseldorf, Germany. The retromer complex in yeast and mammals *Author for correspondence ([email protected]) In yeast, the core retromer complex is composed of three proteins:

R.I., 0000-0001-7909-4123 vacuolar protein sorting 35 (Vps35p), Vps29p and Vps26p (also Journal of Cell Science

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A SNX dimer Core retromer MVB VPS26 VPS29 VPS35 SNX1 SNX2

Vacuole B 302 AtVPS26a (At5g53530) AtVPS26b (At4g27690) 303 190 AtVPS29 (At3g47810) 787 AtVPS35a (At2g17790) 790 AtVPS35b (At1g75850) TGN 790 AtVPS35c (At3g51310) Golgi Nucleus C S16 402 AtSNX1 (At5g06140) ER 587 AtSNX2a (At5g58440)

572 AtSNX2b (At5g07120) Key Secretion Endocytosis 706 AtSNX3.1 (At1g15240.1) Degradation Recycling 1020 AtSNX3.2 (At1g15240.2) AtSNX3.3 (At1g15240.3) 1012 Fig. 1. Endomembrane trafficking pathways in plant cells. Proteins leaving the ER pass the Golgi and localize to the TGN, where the pathways towards the 994 cell surface and the vacuole split (dark gray arrows). Proteins destined for AtSNX4 (At2g15900) the vacuole are transported into the MVB. PM material is endocytosed towards 938 the TGN (blue arrow) and sent for vacuolar degradation via the MVB (orange AtSNX5 (At3g48195) arrows). During the early stages of MVB maturation, certain proteins can be retrieved from the vacuolar pathway and be recycled (green arrows). D 1127 Mdm1p (NM_001182466) known as Pep8p). Vps35p functions as the binding factor for 957 membranes of the prevacuolar compartment and is responsible for the HsSNX13 (NM_015132) interaction with the sorting receptors which are to be recycled (Hierro HsSNX14 (AY044865) 886 992 et al., 2007; Seaman et al., 1998). The classical SNX subcomplex is a HsSNX19 (AF395843) Vps5p–Vps17p heterodimer. Its function is to sense and/or induce HsSNX25 (AY601647) 840 membrane curvature through the Bin-Amphiphysin-Rvs (BAR) domains in both proteins (Peter et al., 2004), which drives the Key retromer towards the forming of endosomal tubules. PX SNX1/2-like MPP signature RGS Retromer components exist in all eukaryotes (Cullen and PX19-like Vps35 signature TM helix Korswagen, 2012). In humans (Homo sapiens), homologs to four PXA BAR Arrestin_N signature of the five yeast retromer proteins have been identified. The Vps17p RING9 PXC protein appears to have no direct homolog, whereas Vps5p has two, SNX1 and SNX2 (Haft et al., 1998; Horazdovsky et al., 1997). Loss Arabidopsis of both proteins causes the human retromer to dissociate from the Fig. 2. The retromer and the SNX protein family. (A) The retromer is a pentamer consisting of two subcomplexes: the core retromer, a endosomal membrane and leads to failure in the trafficking of trimer of the subunits VPS26, VPS29 and VPS35 (light to dark blue), and a SNX the mannose 6-phosphate receptors back to the TGN (Rojas et al., heterodimer (yellow). VPS35 and the SNX proteins, but not VPS26 and VPS29 2007). In addition to the BAR domain, sorting nexins are interact with the membrane surface. In yeast and mammals, the VPS29 subunit characterized by the presence of a specific type of PHOX- interacts with the SNX subcomplex (not depicted). In plants, an interaction homology (PX) domain that allows them to bind to membrane between the two subcomplexes has thus far not been demonstrated. (B) The phosphoinositides (Ponting, 1996; Seaman and Williams, 2002; Arabidopsis genome contains two VPS26-encoding genes, one encoding VPS29 and three encoding VPS35. (C) The Arabidopsis SNX protein family Teasdale et al., 2001). The human SNX family has 33 members: 12 consists of six members, named AtSNX1–5, with two isoforms of AtSNX2. The contain a PX and a BAR domain (including SNX1 and SNX2), ten serine 16 residue in AtSNX1, shown to undergo phosphorylation upon auxin only a PX domain, and 11 harbor the PX domain in combination treatment (Zhang et al., 2013), is depicted as S16. The gene encoding AtSNX3 with a variety of other domains (Cullen, 2008). is predicted to produce three different transcripts owing to alternative splicing. Interestingly, the retromer also uses cargo-specific sorting nexins (D) Homologs of Arabidopsis SNX3 and SNX4 in yeast (Mdm1p) and humans beyond the canonical SNX1 and SNX2. For example, SNX3 in a (HsSNX13, HsSNX14, HsSNX19 and HsSNX25). In all cases, the sequence retromer that lacks the BAR-containing SNX proteins is employed accession numbers are shown. The numbers shown at the C-terminus represent the protein length in amino acids. PX, PHOX homology; BAR, for the recycling of the Wntless sorting receptor (Harterink et al., Bin-Amphiphysin-Rvs; PXA, PHOX-associated; PXC, PHOX C-terminal; RING9, 2011; Zhang et al., 2011). Further, a retromer comprising SNX27 Really Interesting New Gene type 9; RGS, regulator of G-protein signaling; MPP,

and the BAR-containing retromer SNX proteins is needed for the metallophosphoesterase, phosphodiesterase signature; TM, transmembrane. Journal of Cell Science

2 REVIEW Journal of Cell Science (2018) 131, jcs203695. doi:10.1242/jcs.203695 delivery of the β2 adrenergic receptor to the PM (Lauffer et al., screen as a trafficking regulator of ACCELERATED CELL DEATH 2010; Temkin et al., 2011). Which type of retromer is utilized is 11 (ACD11) to the PM. In the absence of AtVPS35b, ACD11 thereby determined by sorting signals present in the cytoplasmic mislocalizes to the late endosomal compartments and the vacuole, part of the cargo. Although the actual endosome exit site can be the which suppresses effector-triggered, immunity-related programmed same for different retromers, the rate of accumulation at the exit site cell death (Munch et al., 2015). AtVPS29 is involved in the depends on the type of retromer (Varandas et al., 2016). In yeast, a transport of the triacylglycerol lipase SUGAR-DEPENDENT 1 SNX3-retromer exists as well, and it promotes the recycling of the (SDP1) between the peroxisome and lipid droplets, a newly iron transporter complex Fet3p–Ftr1p (Strochlic et al., 2007). described function of the plant retromer (Thazar-Poulot et al., Therefore, the yeast and mammalian retromer has a modular 2015). This translocation was shown to occur through tubular composition containing a core and a variable, cargo-specific, extensions of the peroxisome. The tubules were fewer and shorter in SNX component. In comparison, evidence suggests that the plant the absence of AtVPS29, correlating with significant delays in retromer can function in the absence of SNX proteins, as discussed SDP1 translocation (Thazar-Poulot et al., 2015). Thus, the plant further below. core retromer has a critical function in plant development and biotic stress responses, as it enables the translocation of transmembrane or The retromer complex and protein sorting in the plant membrane-associated proteins towards their target compartments. endomembrane system Interaction between AtVPS35a, AtVPS29 and AtVPS26a has In the model plant Arabidopsis (Arabidopsis thaliana, At), there are been confirmed by yeast two-hybrid and co-immunoprecipitation three genes coding for a VPS35 homolog (VPS35a, VPS35b and studies (Jaillais et al., 2007). AtVPS35b and AtVPS29 were VPS35c), two for VPS26 (VPS26a and VPS26b) and only one for independently shown to interact by co-immunoprecipitation VPS29 (Jaillais et al., 2007; Shimada et al., 2006; Yamazaki et al., experiments (Yamazaki et al., 2008). It appears that AtVPS35 2008). Additionally, three BAR-domain SNX-encoding genes are binds membranes independently of AtVPS29, but AtVPS29 known: AtSNX1, AtSNX2a and AtSNX2b, that encode homologs of requires AtVPS26 and AtVPS35 to localize to the endosomal yeast Vps5p (Jaillais et al., 2006; Zelazny et al., 2012) (Fig. 2B). membrane (Zelazny et al., 2013). This is in contrast to yeast, where The core retromer has important roles in the development of only Vps26p is needed for membrane association of the core Arabidopsis. The analysis of mutant plants has suggested that complex (Seaman et al., 1998). Current data suggest that the core AtVPS35a and AtVPS35b might have redundant roles, as loss of Arabidopsis retromer might first assemble in the cytosol before it is function of either gene led to no discernible phenotypes (Yamazaki recruited to membranes owing to the interaction of AtVPS35 with et al., 2008). However, the vps35b vps35c double mutant, as well as the Rab7-type small GTPase RABG3f at the endosomal surface. the vps35a vps35b vps35c triple mutant exhibited major The three VPS proteins have been shown to stay in a complex even developmental alterations, including dwarfism and abnormal seed after their detachment from membranes (Zelazny et al., 2013). development (Yamazaki et al., 2008). Similarly, single loss-of- Furthermore, the correct membrane association of AtVPS29 in function mutants for the AtVPS26a or AtVPS26b gene are SNX-BAR loss-of-function plants suggests that the SNX indistinguishable from the wild type, whereas the double mutant, subcomplex is not involved in the membrane recruitment of the as well as vps29 single mutants, show multiple developmental core retromer (Pourcher et al., 2010), which is not the case in yeast defects (Jaillais et al., 2007; Shimada et al., 2006; Zelazny et al., and mammals. In addition, AtVPS35 stability seems to depend on 2013): for example, vps29 mutant plants have severe disturbances in AtVPS29, as vps29 mutants have very low levels of AtVPS35 the distribution of the plant hormone auxin. These defects are protein (Shimada et al., 2006). Therefore, the plant core retromer related to the abnormal trafficking of auxin efflux carriers of the seems to have an assembly and membrane recruitment strategy that PIN-FORMED (PIN) family. In the wild type, PIN transporters is distinct to the one in yeast and mammals. localize in polar domains of the PM, ensuring the directional auxin The topic of the subcellular localization of the Arabidopsis flow in plant organs. In a vps29 mutant background, AtPIN1 and retromer is surrounded by controversy (Robinson and Pimpl, 2014; AtPIN2 have decreased stability (Kleine-Vehn et al., 2008), and the Robinson et al., 2012). Functional fluorescently tagged AtSNX1, coordination of AtPIN1 repolarization during lateral root initiation AtSNX2a, AtSNX2b, AtVPS29 and AtVPS35 proteins were found is strongly affected (Jaillais et al., 2007). vps26 vps29 double to colocalize with MVB markers, but not with the Golgi or TGN mutants, as well as vps35a vps35b vps35c triple mutants show markers in Arabidopsis root meristems (Jaillais et al., 2006, 2007; abnormal trafficking of storage proteins during late seed Kleine-Vehn et al., 2008; Pourcher et al., 2010; Yamazaki et al., development. Here, storage proteins are incorrectly processed 2008; Zelazny et al., 2013), thus resembling the localization of the owing to their partial mistargeting to the extracellular space, yeast retromer. Furthermore, this MVB localization was supported instead of a targeting to the protein storage vacuole (PSV) (Pourcher by recent proteomic studies, as putative retromer components et al., 2010; Yamazaki et al., 2008; Zelazny et al., 2013). Among the purified together with MVB markers (Heard et al., 2015). In three AtVPS35 proteins, AtVPS35a is required for protein transport contrast, immunoelectron microscopy on Arabidopsis roots, as well to the lytic vacuole, whereas AtVPS35b is mainly involved in as fluorescence immunolocalization-based colocalization analysis protein sorting to the PSV in the maturing seed (Nodzynskí et al., in Arabidopsis roots or tobacco BY2 cells revealed that endogenous 2013; Yamazaki et al., 2008). Using the AtVPS29 knockdown AtVPS29 and AtSNX2a localized at the TGN (Niemes et al., mutant mag1-1, it was shown that the core retromer is also 2010b; Stierhof et al., 2013). In vivo imaging of protoplasts responsible for the recycling of VSRs and, consequently, for the expressing fluorescently tagged AtSNX1 and AtSNX2a confirmed delivery of soluble proteins to the lytic vacuole (Kang et al., 2012; these observations (Niemes et al., 2010b). In addition, the Shimada et al., 2006; Yamazaki et al., 2008). Indeed, a successful localization of AtSNX1–GFP to the TGN was shown by Stierhof co-immunoprecipitation of the Arabidopsis VSR AtBP80 using et al. (2013) in immunoelectron microscopy studies using AtVPS35-specific antibodies has been described (Oliviusson et al., Arabidopsis roots of the same transgenic lines used in Jaillais 2006). Two recent studies have highlighted additional functions of et al. (2006). Furthermore, an AtSNX1–GFP fusion colocalized retromer components. AtVPS35b was identified in a suppressor with both TGN and MVB markers in tobacco epidermis cells Journal of Cell Science

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(Ivanov et al., 2014). In an attempt to consolidate these data, the of VSR-dependent proteins, but it affected their delivery rate to the current models propose a function of the Arabidopsis retromer vacuole (Niemes et al., 2010b). Interestingly, the de novo components both in protein sorting at the TGN and during the early synthesized YFP–BP80 remained at the ER in protoplasts when stages of MVB formation, before their full maturation (Brumbarova SNX function is disturbed. This indicates a potential participation of et al., 2015; Niemes et al., 2010b; Robinson and Neuhaus, 2016; SNX in the selectivity of export events from the ER. The soluble Robinson et al., 2012). Thus, the retromer localization might reflect vacuolar proteins were retained in the ER, together with the VSRs, the versatile TGN functions as an early endosome sorting under these conditions (Niemes et al., 2010a) (Fig. 3A). This shows compartment and a source compartment for the MVB. that SNX proteins might be involved in several stages of VSR trafficking, thus affecting the transport of vacuolar proteins in plant The role of plant sorting nexins in protein trafficking cells. An unresolved issue remains the subunit composition of the plant retromer. The physical interaction between the SNX subcomplex SNX proteins and the trafficking of plant PM-localized and the core retromer is well established in yeast and mammals. In transporters humans, the strength of the interaction differs depending on the snx mutants display only minor defects under standard growth SNX partner (Harterink et al., 2011; Seaman et al., 1998). Despite conditions. However, these plants lack adequate responses when their tight link to the core retromer complex, there is sufficient challenged, for instance in response to environmental stimuli, such evidence that the SNX proteins that participate in the retromer also as gravistimulation, heat or nutrient deficiency (Blum et al., 2014; perform functions independently of it. For example, the recycling of Hanzawa et al., 2013; Ivanov et al., 2014; Jaillais et al., 2006; the G-protein-coupled receptor P2Y1 is unaffected by the depletion Kleine-Vehn et al., 2008; Pourcher et al., 2010). In particular, of VPS26 or VPS35, but strongly increases upon the knockdown of AtSNX1 has been shown to affect the distribution of the the SNX1 gene (Nisar et al., 2010). A similar result had previously phytohormone auxin by controlling the trafficking of the PM been reported for the effect of SNX1 on the degradation of the transporter AtPIN2, which is responsible for the efflux of auxin protease-activated receptor-1 (PAR1) (Gullapalli et al., 2006). As from root epidermis and cortex cells (Jaillais et al., 2006) (Fig. 3B). mentioned above, knockout of Arabidopsis genes encoding core AtPIN2 cycles rapidly between the PM and endosomal retromer components results in severe developmental phenotypes compartments and its targeted depletion from the PM in cells on (Jaillais et al., 2007; Yamazaki et al., 2008; Zelazny et al., 2013), the upper side of the root promotes unequal cell elongation and, whereas even the snx1 snx2a snx2b triple mutant displays only consequently, root bending towards the gravity vector (Abas et al., minor developmental defects under standard growth conditions 2006; Paciorek et al., 2005). Interestingly, an AtPIN2 fusion was (Pourcher et al., 2010). The loss of both AtSNX1 and AtVPS29, shown to colocalize with another AtSNX1 fusion protein in however, leads to embryonic lethality (Jaillais et al., 2007). This endosomes in Arabidopsis root cells (Jaillais et al., 2006). In snx1 supports the notion that the two putative Arabidopsis retromer mutants, the endosomal retrieval of AtPIN2 was compromised and subcomplexes might act separately, but have complementary the transporter was targeted to the vacuole for degradation (Kleine- functions, during plant development. Additionally, at present, Vehn et al., 2008). Consequently, snx mutant plants display altered there is no experimental evidence of a physical interaction between auxin distribution in roots and have problems in realigning root members of the plant core retromer and any of the three Arabidopsis growth in the direction of gravity (Jaillais et al., 2006; Pourcher SNX-BAR proteins. et al., 2010). AtSNX1-dependent endosomal recycling of AtPIN2 is controlled by additional abiotic factors, such as temperature and Sorting nexins and the trafficking of plant vacuolar proteins light. Indeed, AtPIN2 was found to accumulate in the vacuole lumen Mature snx mutant seeds accumulate uncleaved 12S globulin in the dark at standard temperatures (23°C), but is recycled to the precursors and have a reduced vacuole size, an indication of PM in an AtSNX1-dependent manner when the temperature is compromised storage protein delivery to the PSV (Pourcher et al., elevated to 29°C. Thus, auxin homeostasis is adjusted in response to 2010). Such an effect was not observed in the snx2b mutant, but is the environmental conditions (Hanzawa et al., 2013). We also pronounced in snx double and triple mutants. In the seeds of the discovered a role for AtSNX1 in the regulation of iron homeostasis snx1 snx2a snx2b triple mutant, the amounts of uncleaved 12S upon iron deficiency as it modulates the recycling of the iron precursors were comparable to those seen in the vps29 mutant, transporter AtIRT1 (Ivanov et al., 2014). AtIRT1 was shown to which had severe developmental defects. Interestingly, trafficking localize predominantly to endosomes and to cycle rapidly between of 2S albumins, the other major storage protein type in Arabidopsis, endosomes and the PM, where it imports ferrous iron (Barberon is unaffected in snx mutants, in contrast to in vps29 plants (Pourcher et al., 2011). We found that fusion proteins of AtSNX1 and et al., 2010). This suggests a certain degree of specialization of the AtIRT1 partially colocalized in a subpopulation of endosomal SNX proteins in the trafficking of globulins towards the PSV. compartments that corresponded to the TGN (Ivanov et al., 2014). However, it remains unclear how this is connected with the sorting Furthermore, analysis of AtIRT1 stability in plants with abrogated of VSRs (Fig. 3A). AtSNX1 and AtSNX2 affect the trafficking of SNX function suggests that AtSNX1 promotes the recycling of fluorescently labeled BP80, a marker that consists of a luminally AtIRT1 at the TGN, because in the absence of AtSNX1, the localized fluorescent protein fused to the transmembrane and transporter is sent for degradation (Ivanov et al., 2014). We propose cytosolic domains of the pea (Pisum sativum) VSR BP80 (Niemes that only AtSNX2b cooperates with AtSNX1 in this process, since et al., 2010b). A YFP-BP80 fusion was also shown to colocalize immunoprecipitation experiments suggest that AtSNX1 is with AtSNX1 in Arabidopsis root cells (Jaillais et al., 2008). predominantly found in a heterodimer with either AtSNX2a or Truncated forms of AtSNX1 and AtSNX2a designed to prevent AtSNX2b (Pourcher et al., 2010). Furthermore, the expression potential retromer assembly or membrane deformation led to pattern of the AtSNX2a gene does not appear to be compatible with a dramatic changes of the GFP–BP80 localization in protoplasts. In role in iron acquisition (Ivanov et al., 2014). The activity of AtSNX1 the presence of these non-functional SNXs, the marker accumulated also affects the expression pattern of the AtIRT1 gene under abnormally in the TGN. This did not prevent the vacuolar targeting conditions of iron deficiency, which might be connected to its effect Journal of Cell Science

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A Key Fig. 3. Role of SNXs in protein trafficking in Secretion Vacuolar Arabidopsis Recycling proteins . (A) Role of SNX proteins in the trafficking of plant vacuolar sorting receptors (VSR). VSRs interact with soluble vacuole-targeted cargo in the ER lumen. AtSNX1, AtSNX2a and AtSNX2b VSR SNX1 (only SNX1 is represented, green–yellow shape) are PM Vacuole proposed to promote the ER exit of VSR–cargo complexes. At the TGN/MVB transition, the VSR– TGN cargo complex dissociates. The cargo is then MVB VSR transported towards the vacuole, whereas the VSRs are recycled (green arrows) in a SNX-dependent VSR VSR manner. Note that not all VSRs require SNX for their VSR recycling. (B) SNX-dependent recycling of Arabidopsis PM- Nucleus VSR localized transporters AtPIN2 and AtIRT1. Once VSR these transporters have gone through the secretory pathway, their subsequent PM localization depends Golgi ER on repeated cycles of endocytosis (blue arrow) and recycling (green arrow). B PM

Vacuole SNX1

MVB

TGN

Nucleus

ER Golgi

Key Secretion Endocytosis PPM-localized Degradation Recycling ttransporter on auxin homeostasis (Blum et al., 2014; Ivanov et al., 2014), can interact with additional potential target proteins; however, the role Fig. 3B). of this interaction currently is not clear. Thus, SNX proteins are involved in the recycling of specific PM transporters at the TGN, promoting their retargeting to the PM. Regulation of SNX protein function An important open question is whether Arabidopsis SNX proteins SNX proteins are thought to bind phosphoinositides – a function affect their targets directly. Within the yeast retromer, the SNX primarily dependent on the PX domain. As the distribution of the proteins Vps5p and Vps17p are considered to help deform the different phosphoinositides is organelle specific, the affinity of PX membrane; cargo recognition is achieved by Vps35p. However, domains to different phosphoinositides ensures the correct subcellular human SNX1 was discovered owing to its capacity to bind the kinase distribution of SNX proteins (Teasdale and Collins, 2012). Indirect domain of the epidermal growth factor receptor (EGFR) (Haft et al., evidence based on the release of SNX1–GFP from the endosomal 1998; Kurten et al., 1996). Similarly, the first plant sorting nexin membrane after chemical inhibition of phosphatidylinositol 3- described, the Brassica oleracea (Bo)SNX1, was identified in a phosphate [PtdIns(3)P] synthesis suggests that AtSNX1 is able to screen for interactors to the kinase domain of the S-RECEPTOR bind PtdIns(3)P (Pourcher et al., 2010). This was recently confirmed KINASE29 (BoSRK29) (Vanoosthuyse et al., 2003), which is in liposome-binding studies, which, in addition, also showed a responsible for self-pollen recognition during the Brassicaceae self- strong binding of AtSNX1 to PtdIns(3,5)P2 (Hirano et al., 2015). incompatibility response (Ivanov et al., 2010). The recognition event Furthermore, lipid-overlay assays showed that in vitro-expressed occurs at the PM; however, the BoSRK29 homolog BoSRK3 was AtSNX2b has a strong affinity for PtdIns(3)P (Phan et al., 2008). found to prominently localize in BoVPS29-labeled endosomes, Importantly, in the absence of the phosphatidylinositol 3-phosphate suggesting that BoSNX1, or potentially the retromer, might be 5-kinase FORMATION OF APLOID AND BINUCLEATE CELLS involved in SRK trafficking (Ivanov and Gaude, 2009). BoSNX1 was 1 (AtFAB1A), AtSNX1 was predominantly localized in the additionally found to interact with the kinase domains of several cytoplasm, which affected the trafficking of AtPIN2 (Hirano et al., Arabidopsis receptor kinases, but its function in the trafficking of 2015) (Fig. 4). In plant cells, AtSNX1 was able to interact with these receptors has not been investigated (Vanoosthuyse et al., 2003). endosomal membranes on its own, which involved the conserved In summary, plant SNXs have retromer-independent functions in RRY motif within the PX domain (Pourcher et al., 2010). endomembrane protein trafficking. In addition to their role in protein Homodimerization of AtSNX1 required a functional PX domain. recycling at the TGN, they are involved in the ER exit of VSRs. SNX An AtSNX1 version with a mutated PX domain localized to the Journal of Cell Science

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of lysosome-related organelles complex 1 (BLOC-1) subunits 1 and 2, AtBLOS1 and AtBLOS2, the homologs of two subunits of the MC9 mammalian BLOC-1 complex (Cui et al., 2010). In mammalian SNX1PXPX ** SNX1PXPX ** SNX2 cells, the BLOC-1 complex is responsible for vesicle trafficking from endosomes to lysosomes (Li et al., 2007; Raposo et al., 2007). AtBLOS1 depletion leads to increased accumulation of both CLASP * AtPIN1 and AtPIN2 at the PM, suggesting that the BLOS FAB1A * NHX6 BLOS1/2 complex in plants might have a similar function in transport from SNX1 recycling PXPX ** SNX2 degradation endosomes to vacuoles (Cui et al., 2010) (Fig. 4). The role of AtSNX proteins in this process presently is not clear. Second, binding of AtSNX1 to the C-terminal domain of the TGN-localized Na+/H+ antiporter AtNHX6 has been demonstrated. AtNHX6 is required for the trafficking of seed storage proteins during late seed Differential phosphorylation events: development (Ashnest et al., 2015). At present, it is not clear * Auxin accumulation whether AtNHX6 is required for AtSNX1 function, or whether * Ionizing radiation AtNHX6 is cargo that needs to be recycled to a compartment that Osmotic stress * reflects an earlier stage of TGN maturation. In any case, no apparent Fig. 4. Regulation of AtSNX function. AtSNX1 (labeled as SNX1) exists as change of AtNHX6 localization was observed in a snx1 mutant both a cytoplasmic and membrane-bound protein. In the cytosol, it may be background (Ashnest et al., 2015). targeted to proteolytic degradation by MC9 or other proteases. The activity of We recently suggested that the protein partners of AtSNX1 may the SNX1 interactors, 1-phosphatidylinositol-3-phosphate 5-kinase FAB1A help in the regulation of its activity in response to stress and the microtubule-associated protein CLASP, is required for SNX1 (Brumbarova and Ivanov, 2016). We found AtSNX1 itself to be membrane association. Together, FAB1A, CLASP and the NHX6 transporter, transcriptionally upregulated under iron deficiency, and that the also a SNX1-interacting protein, promote endosomal protein recycling. SNX1 can additionally interact with the BLOS1 and BLOS2 proteins, which are AtSNX1 protein is a target for post-translational control. AtSNX1 involved in the vacuolar targeting of endosome-localized proteins. However, has been identified as a target of the proteolytic enzyme the role of this interaction is not entirely clear. Color-coded asterisks indicate METACASPASE 9 (MC9) (Fig. 4), but the significance of this is the known cases where SNX1 or any of its interactors undergoes differential not clear yet. MC9 is exclusively expressed in developing tracheary phosphorylation in response to external stress (summarized in Brumbarova elements (Tsiatsiani et al., 2013), together with genes encoding and Ivanov, 2016). other AtSNX1 interactors, such as BLOS2, CLASP and NHX6 (Brumbarova and Ivanov, 2016); however, the function of SNX1 cytoplasm and failed to either homo- or hetero-dimerize, consistent has not been investigated in these cells. In addition, higher amounts with the observation that AtSNX1 homodimerization occurs of AtSNX1 phosphorylated at serine 16, which is in close proximity exclusively at endosomes (Pourcher et al., 2010). to the PX domain, were found upon elevated auxin levels (Zhang The membrane loading of AtSNX1 is dependent on its interaction et al., 2013). The overexpression of a phospho-mimetic form of with the microtubule-associated protein CYTOPLASMIC LINKER AtSNX1 resulted in the inhibition of primary root growth and lateral ASSOCIATED PROTEIN (AtCLASP). Accordingly, in clasp loss- root development (Zhang et al., 2013). AtSNX1 interactors, such as of-function mutants, AtSNX1 is predominantly cytoplasmic MC9, NHX6, CLASP and FAB1A also show stress-related changes (Ambrose et al., 2013). The AtCLASP–AtSNX1 interaction at transcriptional or post-transcriptional level. This implies that the occurs through a minimal binding domain that is located between composition and activity of AtSNX1-containing complexes can be the PX and BAR domains of AtSNX1. In clasp mutants, AtPIN2– adjusted to match the demands of the plant under a changing GFP accumulated in the lumen of the lytic vacuoles instead of being environment (Brumbarova and Ivanov, 2016). recycled by AtSNX1-containing endosomes. This indicates that AtCLASP – and microtubules – facilitate AtSNX1-mediated Three novel members of the Arabidopsis SNX family recycling of AtPIN2 (Ambrose et al., 2013) (Fig. 4). Until recently, it was considered that the plant SNX proteins were The membrane association of AtSNX2 proteins is dependent on only of the BAR-domain type. We have identified three additional AtSNX1. In snx1 Arabidopsis mutants, AtSNX2b was localized in Arabidopsis SNX-encoding genes (Zelazny et al., 2012) (Fig. 2C). the cytoplasm, and consequently the interaction between AtSNX2a The products of At1g15240 (AtSNX3) and At2g15900 (AtSNX4) and AtSNX2b was also observed exclusively in the cytoplasm in contain SNX19-like PX domains. Furthermore, they show tobacco epidermis cells (Pourcher et al., 2010). However, homology to, and have a domain composition similar to four overexpression of the phosphatidylinositol AtFAB1A is sufficient human SNX proteins, SNX13, SNX14, SNX19 and SNX25. These to recruit AtSNX2b to the endosome, even in the absence of human proteins are predicted to have two N-terminal helical AtSNX1 (Hirano and Sato, 2016). In addition, the membrane transmembrane (TM) domains, followed by a PX-associated (PXA) attachment of AtSNX2a was found to depend on the messenger domain, a regulator of G-protein signaling (RGS) domain, the PX molecule inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. In plants that domain and a conserved C-terminal domain, hereafter referred to as are defective for the enzyme inositol polyphosphate 5-phosphatase a PXC domain (Fig. 2D). Exceptions are SNX25, which lacks the 13 (5PT13), and thus are considered to be Ins(1,4,5)P3 free, predicted TM domains, and SNX19, lacking the RGS domain. The AtSNX2a was predominantly found in the cytoplasm, despite the Arabidopsis AtSNX3 and AtSNX4 also lack the RGS domain. In presence of AtSNX1 (Chu et al., 2016). Thus, membrane loading of comparison, the product encoded by At3g48195 (AtSNX5) has a PX SNX is a complex and highly regulated process that might have domain and a C-terminal RING9-type Zn-finger domain, and has no implications for the fine-tuning of SNX-based protein recycling. obvious homologs in humans (Fig. 2C). AtSNX1 is involved in two additional interactions with proteins There is little available information on the expression patterns and that affect intracellular trafficking. First, it interacts with biogenesis possible interactions of these three Arabidopsis proteins, and in the Journal of Cell Science

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Mdm1p the same time, SNX-BAR proteins themselves have important roles in protein trafficking and recycling, which underlines their significance, especially in plant responses to environmental stress. ER Vacuole We outline a few of the many open questions concerning the plant retromer and the sorting nexins below. A major future challenge will be to identify the protein environment that enables and fine-tunes retromer- and SNX- Key mediated trafficking. These proteins include both cargo and PXA PXC PX19-like regulatory proteins. It will also be important to understand the RGS TM helix role of the different core retromer subunit isoforms. Do AtVPS35a, AtVPS35b and AtVPS35c provide different cargo selectivities? Fig. 5. The proposed function of yeast Mdm1p. The Mdm1 protein is depicted as a gray line containing the indicated domains (boxes). It is proposed With three AtVPS35 and two VPS26 isoforms, are there six to function in tethering the ER and the vacuole membrane through the different compositions of the core retromer? Alternatively, do transmembrane domains and the attachment of the PX domain, respectively. AtVPS35 isoforms have preference for a specific AtVPS26? The arrows indicate the two membranes approaching each other. Importantly, the identification of additional retromer-interacting proteins will enable a better understanding of the mechanisms of absence of mutants, their function remains unknown. However, data retromer-mediated protein trafficking. This is a key question, from yeast and mammalian homologs might provide hints on the especially in the light of the modest involvement of SNX-BAR possible role of these uncharacterized AtSNXs. The yeast proteins, which – based on the knowledge from yeast and mammals mitochondrial distribution and morphology 1 (Mdm1p), a – should be required for the targeting of the complex towards homolog of SNX13, was recently shown to mediate the formation membrane tubulations. of ER–vacuole contact sites. Here, it serves as a tether through its The knowledge on AtSNX1 interactors has already proven to be ER-anchored TM domains and the PX domain, which contacts the useful in understanding how AtSNX1 activity might be modulated vacuolar membrane (Henne et al., 2015) (Fig. 5). in response to environmental cues. In this respect, however, it will The localization of the human SNXs differs from this and was be important to dissect the actual composition and dynamics of the suggested to depend on the preference of the PX domain for AtSNX1-containing protein complexes. The question of how different phosphoinositides. The PX domains of SNX13 and AtSNX1 regulates trafficking of its target proteins is still open. A SNX19 are able to bind to PtdIns(3)P. This is not the case for the PX direct AtSNX1 interaction with VSRs, AtPIN2 or AtIRT1 has so far domain of SNX14, possibly due to minor differences in the amino not been demonstrated, and it remains a future challenge to find out acid sequence and structure of the binding pocket (Mas et al., 2014). whether they interact directly or through other proteins. Instead, the SNX14 PX domain binds to PtdIns(3,5)P2 on late Finally, an intriguing aspect concerning the plant SNX protein endosome and/or lysosome membranes (Akizu et al., 2015). In family will be to understand whether the three novel proteins, addition, it is also recruited to the PM and is responsible for the AtSNX3, AtSNX4 and AtSNX5 have any related or complementary degradation of its direct interactor, the neuronal 5- functions to those of the SNX-BAR proteins and the core retromer. hydroxytryptamine type 6 receptor (5-HT6R) (Ha et al., 2015). In order to obtain information on this, an initial characterization of Mouse SNX13 has also been implicated in PM receptor recycling loss-of-function mutants and subcellular localization experiments and degradation. In addition, homozygous Snx13 mutant mice will be needed. Of course, at this point it cannot be excluded that displayed embryonic lethality (Zheng et al., 2006). Furthermore, the these novel SNX proteins have roles unrelated to SNX-BARs and inactivation of the zebrafish (Danio rerio) homolog of human and the retromer. mouse SNX13 led to the degradative sorting of the early endosome- localized apoptosis repressor with caspase recruitment domain Acknowledgements (ARC) and severe heart failure due to cardiomyocyte apoptosis (Li We would like to thank Tzvetina Brumbarova for her input during the editing of this manuscript. et al., 2014). A function of human SNX25 in receptor recycling has also been shown; it colocalized with the transforming growth factor Competing interests β (TGF-β) receptor in endosomes and promoted its degradation The authors declare no competing or financial interests. (Hao et al., 2011). Therefore, it appears to be a major task of mammalian RGS-PX proteins to mediate subcellular targeting of Funding receptors. It remains to be seen if this is also the case for the Our work is supported by the Strategic Research Fund at the Heinrich-Heine- UniversitätDüsseldorf, Germany (project SFF-F2014/730-15Ivanov) and the Arabidopsis homologs. It will be interesting to test whether Deutsche Forschungsgemeinschaft through the Collaborative Research Center compartment tethering, as observed in yeast, could represent the 1208 (Project B05). underlying molecular mechanism (Fig. 5). References Outlook Abas, L., Benjamins, R., Malenica, N., Paciorek, T., Wisniewska, J., Moulinier- The modular composition of the yeast and mammalian retromer – Anzola, J. C., Sieberer, T., Friml, J. and Luschnig, C. (2006). Intracellular with a core subcomplex and cargo-specific SNX subcomplex – trafficking and proteolysis of the Arabidopsis auxin-efflux facilitator PIN2 are involved in root gravitropism. Nat. Cell Biol. 8, 249-256. allows for a great versatility in cargo protein trafficking. 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