The retromer, sorting nexins and the plant endomembrane protein … · 2018-01-23 · Protein sorting in the endomembrane system is responsible for the coordination of cellular functions.

REVIEW SPECIAL ISSUE: PLANT CELL BIOLOGY

The retromer, sorting nexins and the plant endomembrane proteintraffickingNicole Heucken and Rumen Ivanov*

ABSTRACTProtein sorting in the endomembrane system is responsible for thecoordination of cellular functions. Plant intracellular trafficking has itsown unique features, which include specific regulatory aspects ofendosomal sorting and recycling of cargo proteins, mediated by theretromer complex. Recent work has led to significant progress inunderstanding the role of Arabidopsis retromer subunits in recyclingvacuolar sorting receptors and plasma membrane proteins. As aconsequence, members of the sorting nexin (SNX) protein family andtheir interaction partners have emerged as critical protein traffickingregulators, in particular with regard to adaptation to environmentalchange, such as temperature fluctuations and nutrient deficiency. Inthis Review, we discuss the known and proposed functions of thecomparatively small Arabidopsis SNX protein family. We review theavailable information on the role of the three Bin-Amphiphysin-Rvs(BAR)-domain-containing Arabidopsis thaliana (At)SNX proteins anddiscuss their function in the context of their potential participation inthe plant retromer complex. We also summarize the role of AtSNX1-interacting proteins in different aspects of SNX-dependent proteintrafficking and comment on the potential function of three novel, as yetunexplored, Arabidopsis SNX proteins.

KEY WORDS: Retromer, Sorting nexin, Protein sorting, Vacuolarsorting receptor, Transporter recycling, Environmental response

IntroductionThe correct distribution of proteins in the endomembrane system iscritical for the maintenance of cellular functions and the survival ofthe organism. In plants, trafficking towards the plasma membrane(PM) or the vacuole is a multistep process occurring through severalintracellular compartments. Transmembrane or soluble luminalproteins are synthesized at the endoplasmic reticulum (ER) andtransported towards the Golgi (Fig. 1). The cis-Golgi cisternaeaccept material from the ER and gradually mature. Ultimately, theyform a new tubular-vesicular structure, the trans-Golgi network(TGN), which contains the ER-derived proteins. In plants, the TGNexists close to the trans-Golgi face, but also as a Golgi-independentcompartment (Kang et al., 2011; Viotti et al., 2010). It is a majorhub where the two transport routes – one leading to the PM and theother to the vacuole – are separated. The TGN fulfills the role of anearly endosome (Dettmer et al., 2006; Lam et al., 2007), and is alsoresponsible for sorting and recycling material to or from the Golgi,the PM and the lytic pathway (Gu et al., 2001; Kunzl et al., 2016;Luo et al., 2015; Paez Valencia et al., 2016; Reguera et al., 2015).Consistent with this, certain TGN-localized protein markers localize

to distinct subdomains of the compartment. This shows that themany functions of the TGN might, to a certain extent, be spatiallyseparated (Bassham et al., 2000; Sanderfoot et al., 2001; Staehelinand Kang, 2008).

The late endosome is a structure with smaller internal (luminal)vesicles, giving it the name multivesicular body (MVB). Fromthe TGN, proteins performing functions in the tonoplast or in thevacuole, or those targeted for vacuolar degradation, pass through theMVB (Fig. 1). The MVB was shown to originate from the TGN(Scheuring et al., 2011), supporting the idea that endomembranecompartments represent continuous stages, rather than static structures(Robinson andNeuhaus, 2016). However, it needs to be noted that othertransport routes are known, such as secretion from the Golgi to the PMbypassing the TGN and direct ER-to-vacuole transport (Crowell et al.,2009; Viotti et al., 2013), whose prominence remains to be determined.

Transport between compartments is bidirectional and proteinsmay undergo retrograde transport towards a preceding traffickingstage (Fig. 1). A form of retrograde transport is the recycling ofvacuolar sorting receptors (VSRs) and PM proteins. In the first case,soluble cargo proteins, such as acid hydrolases, move towards thevacuole owing to their interaction with VSRs already at the ER(Kunzl et al., 2016; Niemes et al., 2010a). The conditions in theTGN lumen promote the dissociation of the complex, and whereasthe soluble cargos proceed with the flow towards the vacuole, theVSRs are transported backwards (recycled) towards the ER (Kunzlet al., 2016; Robinson and Neuhaus, 2016). Similarly, endocytosedPM proteins – such as transporters and transmembrane receptors –can be recycled back to the PM by being actively diverted from thedefault vacuolar degradation pathway (Barberon et al., 2011;Dhonukshe et al., 2007; Ivanov et al., 2014; Kasai et al., 2011; Luoet al., 2015; Viotti et al., 2010). The wealth of often contradictorydata suggests that the sorting events that underlie these recyclingprocesses occur at the TGN and most probably involve the earlystages of MVB maturation (Robinson and Neuhaus, 2016).

The retromer is a key protein complex involved in cargo recyclingand retrograde transport. Its components were identified in screensfor yeast (Saccharomyces cerevisiae) mutants defective in vacuolartrafficking (Paravicini et al., 1992; Seaman et al., 1998). Theretromer consists of two subcomplexes, the core retromer and thesorting nexin (SNX) subcomplex (Fig. 2A). In this Review, wediscuss the role of the retromer complex and SNXs in plant proteinsorting. By drawing comparison to the yeast and mammaliansystems, we outline the common and specific functions of the plantretromer. We further concentrate on the plant SNX protein family,which consists of three previously known and three novel, as yetuncharacterized, proteins, and we discuss their localization,regulation and functions.

The retromer complex in yeast and mammalsIn yeast, the core retromer complex is composed of three proteins:vacuolar protein sorting 35 (Vps35p), Vps29p and Vps26p (also

Institute of Botany, Heinrich-Heine University, Universitatsstrasse 1, 40225Dusseldorf, Germany.

*Author for correspondence ([email protected])

R.I., 0000-0001-7909-4123

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© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs203695. doi:10.1242/jcs.203695

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known as Pep8p). Vps35p functions as the binding factor formembranes of the prevacuolar compartment and is responsible for theinteraction with the sorting receptors which are to be recycled (Hierroet al., 2007; Seaman et al., 1998). The classical SNX subcomplex is aVps5p–Vps17p heterodimer. Its function is to sense and/or inducemembrane curvature through the Bin-Amphiphysin-Rvs (BAR)domains in both proteins (Peter et al., 2004), which drives theretromer towards the forming of endosomal tubules.Retromer components exist in all eukaryotes (Cullen and

Korswagen, 2012). In humans (Homo sapiens), homologs to fourof the five yeast retromer proteins have been identified. The Vps17pprotein appears to have no direct homolog, whereas Vps5p has two,SNX1 and SNX2 (Haft et al., 1998; Horazdovsky et al., 1997). Lossof both proteins causes the human retromer to dissociate from theendosomal membrane and leads to failure in the trafficking ofthe mannose 6-phosphate receptors back to the TGN (Rojas et al.,2007). In addition to the BAR domain, sorting nexins arecharacterized by the presence of a specific type of PHOX-homology (PX) domain that allows them to bind to membranephosphoinositides (Ponting, 1996; Seaman and Williams, 2002;Teasdale et al., 2001). The human SNX family has 33 members: 12contain a PX and a BAR domain (including SNX1 and SNX2), tenonly a PX domain, and 11 harbor the PX domain in combinationwith a variety of other domains (Cullen, 2008).Interestingly, the retromer also uses cargo-specific sorting nexins

beyond the canonical SNX1 and SNX2. For example, SNX3 in aretromer that lacks the BAR-containing SNX proteins is employedfor the recycling of the Wntless sorting receptor (Harterink et al.,2011; Zhang et al., 2011). Further, a retromer comprising SNX27and the BAR-containing retromer SNX proteins is needed for the

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Fig. 1. Endomembrane trafficking pathways in plant cells. Proteins leavingthe ER pass theGolgi and localize to the TGN, where the pathways towards thecell surface and the vacuole split (dark gray arrows). Proteins destined forthe vacuole are transported into the MVB. PMmaterial is endocytosed towardsthe TGN (blue arrow) and sent for vacuolar degradation via the MVB (orangearrows). During the early stages of MVB maturation, certain proteins can beretrieved from the vacuolar pathway and be recycled (green arrows).

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Fig. 2. The retromer and the Arabidopsis SNX protein family. (A) Theretromer is a pentamer consisting of two subcomplexes: the core retromer, atrimer of the subunits VPS26, VPS29 and VPS35 (light to dark blue), and a SNXheterodimer (yellow). VPS35 and the SNX proteins, but not VPS26 and VPS29interact with the membrane surface. In yeast and mammals, the VPS29 subunitinteracts with the SNX subcomplex (not depicted). In plants, an interactionbetween the two subcomplexes has thus far not been demonstrated. (B) TheArabidopsis genome contains two VPS26-encoding genes, one encodingVPS29 and three encoding VPS35. (C) The Arabidopsis SNX protein familyconsists of six members, named AtSNX1–5, with two isoforms of AtSNX2. Theserine 16 residue in AtSNX1, shown to undergo phosphorylation upon auxintreatment (Zhang et al., 2013), is depicted as S16. The gene encoding AtSNX3is predicted to produce three different transcripts owing to alternative splicing.(D) Homologs of Arabidopsis SNX3 and SNX4 in yeast (Mdm1p) and humans(HsSNX13, HsSNX14, HsSNX19 and HsSNX25). In all cases, the sequenceaccession numbers are shown. The numbers shown at the C-terminusrepresent the protein length in amino acids. PX, PHOX homology; BAR,Bin-Amphiphysin-Rvs; PXA, PHOX-associated; PXC, PHOX C-terminal; RING9,Really Interesting New Gene type 9; RGS, regulator of G-protein signaling; MPP,metallophosphoesterase, phosphodiesterase signature; TM, transmembrane.

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delivery of the β2 adrenergic receptor to the PM (Lauffer et al.,2010; Temkin et al., 2011). Which type of retromer is utilized isthereby determined by sorting signals present in the cytoplasmicpart of the cargo. Although the actual endosome exit site can be thesame for different retromers, the rate of accumulation at the exit sitedepends on the type of retromer (Varandas et al., 2016). In yeast, aSNX3-retromer exists as well, and it promotes the recycling of theiron transporter complex Fet3p–Ftr1p (Strochlic et al., 2007).Therefore, the yeast and mammalian retromer has a modular

composition containing a core and a variable, cargo-specific,SNX component. In comparison, evidence suggests that the plantretromer can function in the absence of SNX proteins, as discussedfurther below.

The retromer complex and protein sorting in the plantendomembrane systemIn the model plant Arabidopsis (Arabidopsis thaliana, At), there arethree genes coding for a VPS35 homolog (VPS35a, VPS35b andVPS35c), two for VPS26 (VPS26a and VPS26b) and only one forVPS29 (Jaillais et al., 2007; Shimada et al., 2006; Yamazaki et al.,2008). Additionally, three BAR-domain SNX-encoding genes areknown: AtSNX1, AtSNX2a and AtSNX2b, that encode homologs ofyeast Vps5p (Jaillais et al., 2006; Zelazny et al., 2012) (Fig. 2B).The core retromer has important roles in the development of

Arabidopsis. The analysis of mutant plants has suggested thatAtVPS35a and AtVPS35b might have redundant roles, as loss offunction of either gene led to no discernible phenotypes (Yamazakiet al., 2008). However, the vps35b vps35c double mutant, as well asthe vps35a vps35b vps35c triple mutant exhibited majordevelopmental alterations, including dwarfism and abnormal seeddevelopment (Yamazaki et al., 2008). Similarly, single loss-of-function mutants for the AtVPS26a or AtVPS26b gene areindistinguishable from the wild type, whereas the double mutant,as well as vps29 single mutants, show multiple developmentaldefects (Jaillais et al., 2007; Shimada et al., 2006; Zelazny et al.,2013): for example, vps29mutant plants have severe disturbances inthe distribution of the plant hormone auxin. These defects arerelated to the abnormal trafficking of auxin efflux carriers of thePIN-FORMED (PIN) family. In the wild type, PIN transporterslocalize in polar domains of the PM, ensuring the directional auxinflow in plant organs. In a vps29 mutant background, AtPIN1 andAtPIN2 have decreased stability (Kleine-Vehn et al., 2008), and thecoordination of AtPIN1 repolarization during lateral root initiationis strongly affected (Jaillais et al., 2007). vps26 vps29 doublemutants, as well as vps35a vps35b vps35c triple mutants showabnormal trafficking of storage proteins during late seeddevelopment. Here, storage proteins are incorrectly processedowing to their partial mistargeting to the extracellular space,instead of a targeting to the protein storage vacuole (PSV) (Pourcheret al., 2010; Yamazaki et al., 2008; Zelazny et al., 2013). Among thethree AtVPS35 proteins, AtVPS35a is required for protein transportto the lytic vacuole, whereas AtVPS35b is mainly involved inprotein sorting to the PSV in the maturing seed (Nodzyn ski et al.,2013; Yamazaki et al., 2008). Using the AtVPS29 knockdownmutant mag1-1, it was shown that the core retromer is alsoresponsible for the recycling of VSRs and, consequently, for thedelivery of soluble proteins to the lytic vacuole (Kang et al., 2012;Shimada et al., 2006; Yamazaki et al., 2008). Indeed, a successfulco-immunoprecipitation of the Arabidopsis VSR AtBP80 usingAtVPS35-specific antibodies has been described (Oliviusson et al.,2006). Two recent studies have highlighted additional functions ofretromer components. AtVPS35b was identified in a suppressor

screen as a trafficking regulator of ACCELERATEDCELLDEATH11 (ACD11) to the PM. In the absence of AtVPS35b, ACD11mislocalizes to the late endosomal compartments and the vacuole,which suppresses effector-triggered, immunity-related programmedcell death (Munch et al., 2015). AtVPS29 is involved in thetransport of the triacylglycerol lipase SUGAR-DEPENDENT 1(SDP1) between the peroxisome and lipid droplets, a newlydescribed function of the plant retromer (Thazar-Poulot et al.,2015). This translocation was shown to occur through tubularextensions of the peroxisome. The tubules were fewer and shorter inthe absence of AtVPS29, correlating with significant delays inSDP1 translocation (Thazar-Poulot et al., 2015). Thus, the plantcore retromer has a critical function in plant development and bioticstress responses, as it enables the translocation of transmembrane ormembrane-associated proteins towards their target compartments.

Interaction between AtVPS35a, AtVPS29 and AtVPS26a hasbeen confirmed by yeast two-hybrid and co-immunoprecipitationstudies (Jaillais et al., 2007). AtVPS35b and AtVPS29 wereindependently shown to interact by co-immunoprecipitationexperiments (Yamazaki et al., 2008). It appears that AtVPS35binds membranes independently of AtVPS29, but AtVPS29requires AtVPS26 and AtVPS35 to localize to the endosomalmembrane (Zelazny et al., 2013). This is in contrast to yeast, whereonly Vps26p is needed for membrane association of the corecomplex (Seaman et al., 1998). Current data suggest that the coreArabidopsis retromer might first assemble in the cytosol before it isrecruited to membranes owing to the interaction of AtVPS35 withthe Rab7-type small GTPase RABG3f at the endosomal surface.The three VPS proteins have been shown to stay in a complex evenafter their detachment from membranes (Zelazny et al., 2013).Furthermore, the correct membrane association of AtVPS29 inSNX-BAR loss-of-function plants suggests that the SNXsubcomplex is not involved in the membrane recruitment of thecore retromer (Pourcher et al., 2010), which is not the case in yeastand mammals. In addition, AtVPS35 stability seems to depend onAtVPS29, as vps29 mutants have very low levels of AtVPS35protein (Shimada et al., 2006). Therefore, the plant core retromerseems to have an assembly and membrane recruitment strategy thatis distinct to the one in yeast and mammals.

The topic of the subcellular localization of the Arabidopsisretromer is surrounded by controversy (Robinson and Pimpl, 2014;Robinson et al., 2012). Functional fluorescently tagged AtSNX1,AtSNX2a, AtSNX2b, AtVPS29 and AtVPS35 proteins were foundto colocalize with MVB markers, but not with the Golgi or TGNmarkers in Arabidopsis root meristems (Jaillais et al., 2006, 2007;Kleine-Vehn et al., 2008; Pourcher et al., 2010; Yamazaki et al.,2008; Zelazny et al., 2013), thus resembling the localization of theyeast retromer. Furthermore, this MVB localization was supportedby recent proteomic studies, as putative retromer componentspurified together with MVB markers (Heard et al., 2015). Incontrast, immunoelectron microscopy on Arabidopsis roots, as wellas fluorescence immunolocalization-based colocalization analysisin Arabidopsis roots or tobacco BY2 cells revealed that endogenousAtVPS29 and AtSNX2a localized at the TGN (Niemes et al.,2010b; Stierhof et al., 2013). In vivo imaging of protoplastsexpressing fluorescently tagged AtSNX1 and AtSNX2a confirmedthese observations (Niemes et al., 2010b). In addition, thelocalization of AtSNX1–GFP to the TGN was shown by Stierhofet al. (2013) in immunoelectron microscopy studies usingArabidopsis roots of the same transgenic lines used in Jaillaiset al. (2006). Furthermore, an AtSNX1–GFP fusion colocalizedwith both TGN and MVB markers in tobacco epidermis cells

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(Ivanov et al., 2014). In an attempt to consolidate these data, thecurrent models propose a function of the Arabidopsis retromercomponents both in protein sorting at the TGN and during the earlystages of MVB formation, before their full maturation (Brumbarovaet al., 2015; Niemes et al., 2010b; Robinson and Neuhaus, 2016;Robinson et al., 2012). Thus, the retromer localization might reflectthe versatile TGN functions as an early endosome sortingcompartment and a source compartment for the MVB.

The role of plant sorting nexins in protein traffickingAn unresolved issue remains the subunit composition of the plantretromer. The physical interaction between the SNX subcomplexand the core retromer is well established in yeast and mammals. Inhumans, the strength of the interaction differs depending on theSNX partner (Harterink et al., 2011; Seaman et al., 1998). Despitetheir tight link to the core retromer complex, there is sufficientevidence that the SNX proteins that participate in the retromer alsoperform functions independently of it. For example, the recycling ofthe G-protein-coupled receptor P2Y1 is unaffected by the depletionof VPS26 or VPS35, but strongly increases upon the knockdown ofthe SNX1 gene (Nisar et al., 2010). A similar result had previouslybeen reported for the effect of SNX1 on the degradation of theprotease-activated receptor-1 (PAR1) (Gullapalli et al., 2006). Asmentioned above, knockout of Arabidopsis genes encoding coreretromer components results in severe developmental phenotypes(Jaillais et al., 2007; Yamazaki et al., 2008; Zelazny et al., 2013),whereas even the snx1 snx2a snx2b triple mutant displays onlyminor developmental defects under standard growth conditions(Pourcher et al., 2010). The loss of both AtSNX1 and AtVPS29,however, leads to embryonic lethality (Jaillais et al., 2007). Thissupports the notion that the two putative Arabidopsis retromersubcomplexes might act separately, but have complementaryfunctions, during plant development. Additionally, at present,there is no experimental evidence of a physical interaction betweenmembers of the plant core retromer and any of the three ArabidopsisSNX-BAR proteins.

Sorting nexins and the trafficking of plant vacuolar proteinsMature snx mutant seeds accumulate uncleaved 12S globulinprecursors and have a reduced vacuole size, an indication ofcompromised storage protein delivery to the PSV (Pourcher et al.,2010). Such an effect was not observed in the snx2b mutant, but ispronounced in snx double and triple mutants. In the seeds of thesnx1 snx2a snx2b triple mutant, the amounts of uncleaved 12Sprecursors were comparable to those seen in the vps29 mutant,which had severe developmental defects. Interestingly, traffickingof 2S albumins, the other major storage protein type in Arabidopsis,is unaffected in snxmutants, in contrast to in vps29 plants (Pourcheret al., 2010). This suggests a certain degree of specialization of theSNX proteins in the trafficking of globulins towards the PSV.However, it remains unclear how this is connected with the sortingof VSRs (Fig. 3A). AtSNX1 and AtSNX2 affect the trafficking offluorescently labeled BP80, a marker that consists of a luminallylocalized fluorescent protein fused to the transmembrane andcytosolic domains of the pea (Pisum sativum) VSR BP80 (Niemeset al., 2010b). A YFP-BP80 fusion was also shown to colocalizewith AtSNX1 in Arabidopsis root cells (Jaillais et al., 2008).Truncated forms of AtSNX1 and AtSNX2a designed to preventpotential retromer assembly or membrane deformation led todramatic changes of the GFP–BP80 localization in protoplasts. Inthe presence of these non-functional SNXs, the marker accumulatedabnormally in the TGN. This did not prevent the vacuolar targeting

of VSR-dependent proteins, but it affected their delivery rate to thevacuole (Niemes et al., 2010b). Interestingly, the de novosynthesized YFP–BP80 remained at the ER in protoplasts whenSNX function is disturbed. This indicates a potential participation ofSNX in the selectivity of export events from the ER. The solublevacuolar proteins were retained in the ER, together with the VSRs,under these conditions (Niemes et al., 2010a) (Fig. 3A). This showsthat SNX proteins might be involved in several stages of VSRtrafficking, thus affecting the transport of vacuolar proteins in plantcells.

SNX proteins and the trafficking of plant PM-localizedtransporterssnx mutants display only minor defects under standard growthconditions. However, these plants lack adequate responses whenchallenged, for instance in response to environmental stimuli, suchas gravistimulation, heat or nutrient deficiency (Blum et al., 2014;Hanzawa et al., 2013; Ivanov et al., 2014; Jaillais et al., 2006;Kleine-Vehn et al., 2008; Pourcher et al., 2010). In particular,AtSNX1 has been shown to affect the distribution of thephytohormone auxin by controlling the trafficking of the PMtransporter AtPIN2, which is responsible for the efflux of auxinfrom root epidermis and cortex cells (Jaillais et al., 2006) (Fig. 3B).AtPIN2 cycles rapidly between the PM and endosomalcompartments and its targeted depletion from the PM in cells onthe upper side of the root promotes unequal cell elongation and,consequently, root bending towards the gravity vector (Abas et al.,2006; Paciorek et al., 2005). Interestingly, an AtPIN2 fusion wasshown to colocalize with another AtSNX1 fusion protein inendosomes in Arabidopsis root cells (Jaillais et al., 2006). In snx1mutants, the endosomal retrieval of AtPIN2 was compromised andthe transporter was targeted to the vacuole for degradation (Kleine-Vehn et al., 2008). Consequently, snx mutant plants display alteredauxin distribution in roots and have problems in realigning rootgrowth in the direction of gravity (Jaillais et al., 2006; Pourcheret al., 2010). AtSNX1-dependent endosomal recycling of AtPIN2 iscontrolled by additional abiotic factors, such as temperature andlight. Indeed, AtPIN2was found to accumulate in the vacuole lumenin the dark at standard temperatures (23°C), but is recycled to thePM in an AtSNX1-dependent manner when the temperature iselevated to 29°C. Thus, auxin homeostasis is adjusted in response tothe environmental conditions (Hanzawa et al., 2013). We alsodiscovered a role for AtSNX1 in the regulation of iron homeostasisupon iron deficiency as it modulates the recycling of the irontransporter AtIRT1 (Ivanov et al., 2014). AtIRT1 was shown tolocalize predominantly to endosomes and to cycle rapidly betweenendosomes and the PM, where it imports ferrous iron (Barberonet al., 2011). We found that fusion proteins of AtSNX1 andAtIRT1 partially colocalized in a subpopulation of endosomalcompartments that corresponded to the TGN (Ivanov et al., 2014).Furthermore, analysis of AtIRT1 stability in plants with abrogatedSNX function suggests that AtSNX1 promotes the recycling ofAtIRT1 at the TGN, because in the absence of AtSNX1, thetransporter is sent for degradation (Ivanov et al., 2014). We proposethat only AtSNX2b cooperates with AtSNX1 in this process, sinceimmunoprecipitation experiments suggest that AtSNX1 ispredominantly found in a heterodimer with either AtSNX2a orAtSNX2b (Pourcher et al., 2010). Furthermore, the expressionpattern of the AtSNX2a gene does not appear to be compatible with arole in iron acquisition (Ivanov et al., 2014). The activity of AtSNX1also affects the expression pattern of the AtIRT1 gene underconditions of iron deficiency, which might be connected to its effect

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on auxin homeostasis (Blum et al., 2014; Ivanov et al., 2014),Fig. 3B).Thus, SNX proteins are involved in the recycling of specific PM

transporters at the TGN, promoting their retargeting to the PM.An important open question is whether Arabidopsis SNX proteins

affect their targets directly. Within the yeast retromer, the SNXproteins Vps5p and Vps17p are considered to help deform themembrane; cargo recognition is achieved by Vps35p. However,human SNX1 was discovered owing to its capacity to bind the kinasedomain of the epidermal growth factor receptor (EGFR) (Haft et al.,1998; Kurten et al., 1996). Similarly, the first plant sorting nexindescribed, the Brassica oleracea (Bo)SNX1, was identified in ascreen for interactors to the kinase domain of the S-RECEPTORKINASE29 (BoSRK29) (Vanoosthuyse et al., 2003), which isresponsible for self-pollen recognition during the Brassicaceae self-incompatibility response (Ivanov et al., 2010). The recognition eventoccurs at the PM; however, the BoSRK29 homolog BoSRK3 wasfound to prominently localize in BoVPS29-labeled endosomes,suggesting that BoSNX1, or potentially the retromer, might beinvolved in SRK trafficking (Ivanov andGaude, 2009). BoSNX1wasadditionally found to interact with the kinase domains of severalArabidopsis receptor kinases, but its function in the trafficking ofthese receptors has not been investigated (Vanoosthuyse et al., 2003).In summary, plant SNXs have retromer-independent functions inendomembrane protein trafficking. In addition to their role in proteinrecycling at the TGN, they are involved in the ER exit of VSRs. SNX

can interact with additional potential target proteins; however, the roleof this interaction currently is not clear.

Regulation of SNX protein functionSNX proteins are thought to bind phosphoinositides – a functionprimarily dependent on the PX domain. As the distribution of thedifferent phosphoinositides is organelle specific, the affinity of PXdomains to different phosphoinositides ensures the correct subcellulardistribution of SNX proteins (Teasdale and Collins, 2012). Indirectevidence based on the release of SNX1–GFP from the endosomalmembrane after chemical inhibition of phosphatidylinositol 3-phosphate [PtdIns(3)P] synthesis suggests that AtSNX1 is able tobind PtdIns(3)P (Pourcher et al., 2010). This was recently confirmedin liposome-binding studies, which, in addition, also showed astrong binding of AtSNX1 to PtdIns(3,5)P2 (Hirano et al., 2015).Furthermore, lipid-overlay assays showed that in vitro-expressedAtSNX2b has a strong affinity for PtdIns(3)P (Phan et al., 2008).Importantly, in the absence of the phosphatidylinositol 3-phosphate5-kinase FORMATION OF APLOID AND BINUCLEATE CELLS1 (AtFAB1A), AtSNX1 was predominantly localized in thecytoplasm, which affected the trafficking of AtPIN2 (Hirano et al.,2015) (Fig. 4). In plant cells, AtSNX1 was able to interact withendosomal membranes on its own, which involved the conservedRRY motif within the PX domain (Pourcher et al., 2010).Homodimerization of AtSNX1 required a functional PX domain.An AtSNX1 version with a mutated PX domain localized to the

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Fig. 3. Role of SNXs in protein trafficking inArabidopsis. (A) Role of SNX proteins in thetrafficking of plant vacuolar sorting receptors (VSR).VSRs interact with soluble vacuole-targeted cargo inthe ER lumen. AtSNX1, AtSNX2a and AtSNX2b(only SNX1 is represented, green–yellow shape) areproposed to promote the ER exit of VSR–cargocomplexes. At the TGN/MVB transition, the VSR–cargo complex dissociates. The cargo is thentransported towards the vacuole, whereas the VSRsare recycled (green arrows) in a SNX-dependentmanner. Note that not all VSRs require SNX for theirrecycling.(B) SNX-dependent recycling of Arabidopsis PM-localized transporters AtPIN2 and AtIRT1. Oncethese transporters have gone through the secretorypathway, their subsequent PM localization dependson repeated cycles of endocytosis (blue arrow) andrecycling (green arrow).

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cytoplasm and failed to either homo- or hetero-dimerize, consistentwith the observation that AtSNX1 homodimerization occursexclusively at endosomes (Pourcher et al., 2010).The membrane loading of AtSNX1 is dependent on its interaction

with the microtubule-associated protein CYTOPLASMIC LINKERASSOCIATED PROTEIN (AtCLASP). Accordingly, in clasp loss-of-function mutants, AtSNX1 is predominantly cytoplasmic(Ambrose et al., 2013). The AtCLASP–AtSNX1 interactionoccurs through a minimal binding domain that is located betweenthe PX and BAR domains of AtSNX1. In clasp mutants, AtPIN2–GFP accumulated in the lumen of the lytic vacuoles instead of beingrecycled by AtSNX1-containing endosomes. This indicates thatAtCLASP – and microtubules – facilitate AtSNX1-mediatedrecycling of AtPIN2 (Ambrose et al., 2013) (Fig. 4).The membrane association of AtSNX2 proteins is dependent on

AtSNX1. In snx1 Arabidopsis mutants, AtSNX2b was localized inthe cytoplasm, and consequently the interaction between AtSNX2aand AtSNX2b was also observed exclusively in the cytoplasm intobacco epidermis cells (Pourcher et al., 2010). However,overexpression of the phosphatidylinositol AtFAB1A is sufficientto recruit AtSNX2b to the endosome, even in the absence ofAtSNX1 (Hirano and Sato, 2016). In addition, the membraneattachment of AtSNX2a was found to depend on the messengermolecule inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]. In plants thatare defective for the enzyme inositol polyphosphate 5-phosphatase13 (5PT13), and thus are considered to be Ins(1,4,5)P3 free,AtSNX2a was predominantly found in the cytoplasm, despite thepresence of AtSNX1 (Chu et al., 2016). Thus, membrane loading ofSNX is a complex and highly regulated process that might haveimplications for the fine-tuning of SNX-based protein recycling.AtSNX1 is involved in two additional interactions with proteins

that affect intracellular trafficking. First, it interacts with biogenesis

of lysosome-related organelles complex 1 (BLOC-1) subunits 1 and2, AtBLOS1 and AtBLOS2, the homologs of two subunits of themammalian BLOC-1 complex (Cui et al., 2010). In mammaliancells, the BLOC-1 complex is responsible for vesicle traffickingfrom endosomes to lysosomes (Li et al., 2007; Raposo et al., 2007).AtBLOS1 depletion leads to increased accumulation of bothAtPIN1 and AtPIN2 at the PM, suggesting that the BLOScomplex in plants might have a similar function in transport fromendosomes to vacuoles (Cui et al., 2010) (Fig. 4). The role ofAtSNX proteins in this process presently is not clear. Second,binding of AtSNX1 to the C-terminal domain of the TGN-localizedNa+/H+ antiporter AtNHX6 has been demonstrated. AtNHX6 isrequired for the trafficking of seed storage proteins during late seeddevelopment (Ashnest et al., 2015). At present, it is not clearwhether AtNHX6 is required for AtSNX1 function, or whetherAtNHX6 is cargo that needs to be recycled to a compartment thatreflects an earlier stage of TGNmaturation. In any case, no apparentchange of AtNHX6 localization was observed in a snx1 mutantbackground (Ashnest et al., 2015).

We recently suggested that the protein partners of AtSNX1 mayhelp in the regulation of its activity in response to stress(Brumbarova and Ivanov, 2016). We found AtSNX1 itself to betranscriptionally upregulated under iron deficiency, and that theAtSNX1 protein is a target for post-translational control. AtSNX1has been identified as a target of the proteolytic enzymeMETACASPASE 9 (MC9) (Fig. 4), but the significance of this isnot clear yet.MC9 is exclusively expressed in developing trachearyelements (Tsiatsiani et al., 2013), together with genes encodingother AtSNX1 interactors, such as BLOS2, CLASP and NHX6(Brumbarova and Ivanov, 2016); however, the function of SNX1has not been investigated in these cells. In addition, higher amountsof AtSNX1 phosphorylated at serine 16, which is in close proximityto the PX domain, were found upon elevated auxin levels (Zhanget al., 2013). The overexpression of a phospho-mimetic form ofAtSNX1 resulted in the inhibition of primary root growth and lateralroot development (Zhang et al., 2013). AtSNX1 interactors, such asMC9, NHX6, CLASP and FAB1A also show stress-related changesat transcriptional or post-transcriptional level. This implies that thecomposition and activity of AtSNX1-containing complexes can beadjusted to match the demands of the plant under a changingenvironment (Brumbarova and Ivanov, 2016).

Three novel members of the Arabidopsis SNX familyUntil recently, it was considered that the plant SNX proteins wereonly of the BAR-domain type. We have identified three additionalArabidopsis SNX-encoding genes (Zelazny et al., 2012) (Fig. 2C).The products of At1g15240 (AtSNX3) and At2g15900 (AtSNX4)contain SNX19-like PX domains. Furthermore, they showhomology to, and have a domain composition similar to fourhuman SNX proteins, SNX13, SNX14, SNX19 and SNX25. Thesehuman proteins are predicted to have two N-terminal helicaltransmembrane (TM) domains, followed by a PX-associated (PXA)domain, a regulator of G-protein signaling (RGS) domain, the PXdomain and a conserved C-terminal domain, hereafter referred to asa PXC domain (Fig. 2D). Exceptions are SNX25, which lacks thepredicted TM domains, and SNX19, lacking the RGS domain. TheArabidopsis AtSNX3 and AtSNX4 also lack the RGS domain. Incomparison, the product encoded by At3g48195 (AtSNX5) has a PXdomain and a C-terminal RING9-type Zn-finger domain, and has noobvious homologs in humans (Fig. 2C).

There is little available information on the expression patterns andpossible interactions of these three Arabidopsis proteins, and in the

PX PXXPXPPXPXPX PXPPSNX1 SNX2 recycling degradation

PX PXXXXXPXPPXPPXPXXPPXX SNX1 SNX2

NHX6 FAB1A CLASP

BLOS1/2

MC9

** * *

**

Differential phosphorylation events: * Auxin accumulation * Ionizing radiation * Osmotic stress

PX PXXXXXPXPPXPPXPXXPPXX SNX1 **

Fig. 4. Regulation of AtSNX function. AtSNX1 (labeled as SNX1) exists asboth a cytoplasmic and membrane-bound protein. In the cytosol, it may betargeted to proteolytic degradation by MC9 or other proteases. The activity ofthe SNX1 interactors, 1-phosphatidylinositol-3-phosphate 5-kinase FAB1Aand the microtubule-associated protein CLASP, is required for SNX1membrane association. Together, FAB1A, CLASP and the NHX6 transporter,also a SNX1-interacting protein, promote endosomal protein recycling. SNX1can additionally interact with the BLOS1 and BLOS2 proteins, which areinvolved in the vacuolar targeting of endosome-localized proteins. However,the role of this interaction is not entirely clear. Color-coded asterisks indicatethe known cases where SNX1 or any of its interactors undergoes differentialphosphorylation in response to external stress (summarized in Brumbarovaand Ivanov, 2016).

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absence of mutants, their function remains unknown. However, datafrom yeast and mammalian homologs might provide hints on thepossible role of these uncharacterized AtSNXs. The yeastmitochondrial distribution and morphology 1 (Mdm1p), ahomolog of SNX13, was recently shown to mediate the formationof ER–vacuole contact sites. Here, it serves as a tether through itsER-anchored TM domains and the PX domain, which contacts thevacuolar membrane (Henne et al., 2015) (Fig. 5).The localization of the human SNXs differs from this and was

suggested to depend on the preference of the PX domain fordifferent phosphoinositides. The PX domains of SNX13 andSNX19 are able to bind to PtdIns(3)P. This is not the case for the PXdomain of SNX14, possibly due to minor differences in the aminoacid sequence and structure of the binding pocket (Mas et al., 2014).Instead, the SNX14 PX domain binds to PtdIns(3,5)P2 on lateendosome and/or lysosome membranes (Akizu et al., 2015). Inaddition, it is also recruited to the PM and is responsible for thedegradation of its direct interactor, the neuronal 5-hydroxytryptamine type 6 receptor (5-HT6R) (Ha et al., 2015).Mouse SNX13 has also been implicated in PM receptor recyclingand degradation. In addition, homozygous Snx13 mutant micedisplayed embryonic lethality (Zheng et al., 2006). Furthermore, theinactivation of the zebrafish (Danio rerio) homolog of human andmouse SNX13 led to the degradative sorting of the early endosome-localized apoptosis repressor with caspase recruitment domain(ARC) and severe heart failure due to cardiomyocyte apoptosis (Liet al., 2014). A function of human SNX25 in receptor recycling hasalso been shown; it colocalized with the transforming growth factorβ (TGF-β) receptor in endosomes and promoted its degradation(Hao et al., 2011). Therefore, it appears to be a major task ofmammalian RGS-PX proteins to mediate subcellular targeting ofreceptors. It remains to be seen if this is also the case for theArabidopsis homologs. It will be interesting to test whethercompartment tethering, as observed in yeast, could represent theunderlying molecular mechanism (Fig. 5).

OutlookThe modular composition of the yeast and mammalian retromer –with a core subcomplex and cargo-specific SNX subcomplex –allows for a great versatility in cargo protein trafficking. Incomparison, the plant retromer shows several unique features,which makes the understanding of its molecular function a rathercomplicated task. All components of the core retromer and the BARdomain-containing SNX proteins are present in plants. However,genetic analysis in Arabidopsis suggests that the SNX-BARproteins are not required for the majority of retromer functions. At

the same time, SNX-BAR proteins themselves have important rolesin protein trafficking and recycling, which underlines theirsignificance, especially in plant responses to environmental stress.We outline a few of the many open questions concerning the plantretromer and the sorting nexins below.

A major future challenge will be to identify the proteinenvironment that enables and fine-tunes retromer- and SNX-mediated trafficking. These proteins include both cargo andregulatory proteins. It will also be important to understand therole of the different core retromer subunit isoforms. Do AtVPS35a,AtVPS35b and AtVPS35c provide different cargo selectivities?With three AtVPS35 and two VPS26 isoforms, are there sixdifferent compositions of the core retromer? Alternatively, doAtVPS35 isoforms have preference for a specific AtVPS26?Importantly, the identification of additional retromer-interactingproteins will enable a better understanding of the mechanisms ofretromer-mediated protein trafficking. This is a key question,especially in the light of the modest involvement of SNX-BARproteins, which – based on the knowledge from yeast and mammals– should be required for the targeting of the complex towardsmembrane tubulations.

The knowledge on AtSNX1 interactors has already proven to beuseful in understanding how AtSNX1 activity might be modulatedin response to environmental cues. In this respect, however, it willbe important to dissect the actual composition and dynamics of theAtSNX1-containing protein complexes. The question of howAtSNX1 regulates trafficking of its target proteins is still open. Adirect AtSNX1 interaction with VSRs, AtPIN2 or AtIRT1 has so farnot been demonstrated, and it remains a future challenge to find outwhether they interact directly or through other proteins.

Finally, an intriguing aspect concerning the plant SNX proteinfamily will be to understand whether the three novel proteins,AtSNX3, AtSNX4 and AtSNX5 have any related or complementaryfunctions to those of the SNX-BAR proteins and the core retromer.In order to obtain information on this, an initial characterization ofloss-of-function mutants and subcellular localization experimentswill be needed. Of course, at this point it cannot be excluded thatthese novel SNX proteins have roles unrelated to SNX-BARs andthe retromer.

AcknowledgementsWe would like to thank Tzvetina Brumbarova for her input during the editing of thismanuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingOur work is supported by the Strategic Research Fund at the Heinrich-Heine-Universitat Dusseldorf, Germany (project SFF-F2014/730-15Ivanov) and theDeutsche Forschungsgemeinschaft through the Collaborative Research Center1208 (Project B05).

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