BACKGROUND
auxin plays important roles in plant growth and development.
directional cell-to-cell transport and the formation of concentration gradients of auxin are pivotal for its biological function.
diffusive cellular auxin efflux is not effective because of ionization of intracellular auxin, so asymmetrically localized auxin efflux carriers in the plasma membrane  play a critical role in polar auxin transport  <cit> .
pin-formed  auxin efflux carrier proteins show a distinctive asymmetrical subcellular distribution, which is dynamically regulated by environmental and developmental cues, and their mutations cause defects in auxin gradient formation and plant growth and development  <cit> .
the arabidopsis pin family consists of eight members, among which six pins  have a long central hydrophilic loop  connecting the five transmembrane  helices on each end.
these ‘long’ pins mainly localize to the pm.
conversely, the other two pins  have a very short  hl and localize to the internal compartment  or to both the internal compartment and the pm   <cit> .
long pins show diverse polar localization in the pm of diverse cell types  <cit> .
although de novo synthesized long pins symmetrically localize to the pm, subsequent cycles of endocytosis and directional recycling  processes determine and maintain specific pin polarity  <cit> .
the endocytosis/recycling process of long pins is coordinated and regulated by environmental and cellular signals  <cit> .
protein phosphorylation and dephosphorylation have been implicated in auxin transport and pin trafficking  <cit> .
there is no direct evidence of a role for the tm domain in auxin transport by pins, but several lines of evidence support the idea that the hl acts in regulation of intracellular trafficking of pin proteins.
the hl of long pins contains several phosphorylation motifs that are targeted by some kinases.
protein kinase inhibitors suppressed cellular auxin transport  <cit> , and interfered with the trafficking of pin proteins to the pm  <cit> .
mutation or overexpression of pinoid  changed auxin transport activity and led to phenotypes associated with auxin transport defects  <cit> .
it has been proposed that changes in phosphorylation status by the antagonistic activities between pid  and the protein phosphatase pp2a modulate the subcellular polarity of pins  <cit> .
accordingly, it has been demonstrated that the pin-hl can be phosphorylated by pid and related kinases in vitro and in a protoplast phosphorylation assay, and that the phosphorylation sites of the pin-hl are required for pins’ polarity and biological functions  <cit> .
recently, we reported a phosphorylatable motif, named m <dig>  in the pin3-hl that includes five s/t  residues and is necessary for the polarity and intracellular trafficking of pin <dig>  <cit> .
root hair-specific overexpression of wild-type pin <dig> greatly inhibited root hair growth by exporting auxin from the root hair cell, whereas overexpression of a phosphorylation-defective m <dig> mutant pin <dig> failed to inhibit root hair growth.
to restore root hair growth, at least three phosphorylation-defective mutations among the five s/t residues were required.
the pm localization of pin <dig> in root hair cells was disrupted and internalized by the phosphorylation-defective mutations because of the defect in pm targeting.
the effect of m <dig> phosphorylation-defective mutation on the subcellular localization of pin <dig> was also observed in its native expression domain, root pericycle cells, resulting in aberrant localization to the outer lateral pm and defects in gravitropism.
the m <dig> motif sequence is conserved among all long pins.
in this study, using the root hair assay system for pin activity, we investigated whether this motif has been functionally conserved among long pins.
RESULTS
to examine the functional conservation of the m <dig> motif among long pins, we chose pin <dig>  and two other well-studied long pins, pin <dig> and pin <dig>  the five phosphorylatable s/t residues in the m <dig> motif are strictly conserved with very few exceptions among long pins including pin <dig>   <dig>  and  <dig> .
most of the m <dig> s/t residues from pin <dig>   <dig>  and  <dig> are predicted to be phosphorylatable by netphos  <dig>  , as are the pin <dig> residues .
pin <dig> shows a unique feature in that its m <dig> motif lacks one putative phosphorylation serine that is replaced by alanine .
while the m <dig> phosphorylation sites are conserved, there is diversity in the sequence between the phosphorylation sites.
these features of m <dig> in long pins indicate both conservation and probably specific divergence in function.
the m <dig> motif is required for the auxin efflux activity of pin <dig>  pin <dig>  and pin <dig> in the arabidopsis root hair cell
in a previous study, we divided the m <dig> s/t sites into two sub-motifs, 3 m <dig> comprising the first three s residues and 2 m <dig> comprising the last two t/s residues .
mutation of the 3 m1 s residues caused defects in the phosphorylation, trafficking, efflux activity, and biological functions of pin <dig>  but 2 m <dig> mutation did not  <cit> .
to assess the function of the m <dig> motif in the auxin efflux activity of pin <dig>  pin <dig>  and pin <dig>  we generated phosphorylation-defective  m <dig>  and 3 m <dig>  mutant pin:gfp  fusion constructs, which were root-hair specifically expressed under the expansin a <dig> promoter  in arabidopsis plants.
as previously reported, this root hair system was adopted to indirectly estimate the pins’ auxin efflux activity because root hair growth depends on auxin levels in the root hair cell  <cit> .
consistent with our previous report  <cit> , root hair-specific overexpression of wild-type pin <dig>  pin <dig>  or pin <dig> greatly inhibited the root hair growth of 4-d-old seedlings by depleting auxin levels in the root hair cell .
on the other hand, this pin-mediated root hair inhibition was significantly suppressed by 3 m <dig> or m <dig> mutation, though m <dig> mutation consistently showed a greater restoration of root hair growth than did 3 m <dig> mutation , similarly to previous results with pin <dig>  <cit> .
among the three pins, the 3 m <dig> and m <dig> mutants of pin <dig> showed the greatest restoration of root hair growth .
these results were reproducible with  <dig> independent transgenic lines for each pin .
in the case of pin <dig>  3 m <dig> and m <dig> mutations suppressed the auxin efflux activity, and thus root hair inhibition, of pin <dig> by interrupting its trafficking to the pm  <cit> .
to test whether this was the same for pin <dig>  pin <dig>  and pin <dig>  we examined the subcellular localization of the mutant forms of these pins.
the m <dig> motif is required for pm targeting of pin <dig> and pin <dig> in the root hair cell
to determine whether the mutant pins were pm or internally localized, gfp-tagged pins were first co-visualized with the endocytic tracer fm4- <dig> in the root hair cell.
wild-type pin <dig> and pin <dig> overlapped with the fm4- <dig> signal at the pm, whereas the 3 m1- and m3-mutant forms of pin <dig> and pin <dig> showed considerable internal localization, though they were partly localized in the pm as well .
this internal localization pattern of mutated pin <dig> and pin <dig> was consistently observed in  <dig> independent transgenic lines .
observation of two representative transgenic lines of 3 m1- and m3-pins showed that pin <dig> internalization occurred in ~50% of the root hair cells, whereas, in the case of pin <dig>  internalization occurred in ~30% of 3 m <dig> and ~55% of m <dig> mutant transgenic lines under similar conditions .
these data support the idea that the probable phosphorylation-defective mutations in the 3 m <dig> or m <dig> motifs significantly impaired the capability of pin <dig> and pin <dig> to target to the pm and thus resulted in suppression of pin-mediated root hair inhibition.
the exocytosis inhibitor brefeldin a  causes internal accumulation of pin proteins into so-called ‘bfa compartments’, which are indicative of pm-localization of pins  <cit> .
to clarify the subcellular localization of mutant pin <dig> and pin <dig> proteins, the transgenic seedlings were co-treated with the protein synthesis inhibitor cycloheximide  and bfa.
signals of wild-type pin <dig> and pin <dig> proteins completely overlapped with those of fm4- <dig> in the bfa compartments, whereas 3 m <dig> or m <dig> mutant pin <dig> and pin <dig> proteins only partly co-localized with the fm4-64-stained bfa compartments .
the lack of internal mutant pin signal and bfa compartment overlap further supports the idea that the ability of pin <dig> and pin <dig> to target to the pm was partially disrupted by the 3 m <dig> and m <dig> phosphorylation-defective mutations.
m <dig> mutations lead to rapid degradation of pin <dig> through the vacuolar lytic pathway in the root hair cell
unlike phosphorylation-defective pin <dig> and pin <dig>  the mutant pin <dig> proteins showed completely different trafficking behavior.
while wild-type pin <dig> was clearly localized to the pm, phosphorylation-defective 3 m1- or m3-pin <dig> proteins were barely detectable in root hair cells; this was consistent in all five independent transgenic lines observed for each mutant construct .
this result was in accordance with the results from the root-hair-inhibition assay, where 3 m <dig> or m <dig> mutant pin2s showed much weaker inhibitory effects  on root hair growth than did mutant pin <dig> or pin <dig> .
the very low expression of these mutant pin <dig> proteins in the cell could be due to either translational failure or degradation of the mutant proteins because the mutant transgenes seemed to be normally transcribed, as shown in the rt-pcr analysis .
because there were some residual inhibitory effects on root hair growth as mentioned above, it is conceivable that a certain portion of mutant pin <dig> proteins might stay transiently in the pm, resulting in weak auxin-exporting activity, and then undergo endocytosis and degradation.
to test this idea and to restore the mutant pin <dig> protein in the cell, we applied auxin  to inhibit clathrin-dependent pin endocytosis from the pm  <cit> , mg <dig> to inhibit ubiquitylation/proteasome-mediated pin internalization and degradation  <cit> , and wortmannin to inhibit the vacuolar lytic pathway  <cit> .
naa  did not have much effect on the pm-localization of wild-type pin <dig> or restored 3 m1- or m3-pin <dig> proteins in the root hair cell .
mg <dig>  caused partial internal accumulation of wild-type pin <dig> proteins, but failed to recover the mutant pin <dig> signals in the root hair cell .
wortmannin, an inhibitor of phosphatidylinositol-3-kinase, inhibits the trafficking of pin proteins as well as fm4- <dig> to the vacuole  <cit> .
wortmannin treatment  restored the gfp signals of 3 m <dig> and m <dig> mutant pin2:gfp proteins in the root hair cell .
when five independent lines for each of two mutant pin <dig> constructs were observed, the intensities of wortmannin-restored mutant pin <dig> signals were similar to those of wild-type pin <dig> , implying that the translational efficiency of the wild-type and mutant pin2s was similar, but that the 3 m <dig> or m <dig> phosphor-defective pin <dig> proteins tended to enter the lytic pathway more than the wild-type protein.
discussion
in this study, we investigated whether the m <dig> motif, a phosphorylation motif originally found in the pin3-hl that regulates pin3’s polarity and intracellular trafficking  <cit> , has been functionally conserved in other long-looped pins using the root hair assay system to estimate pin activity and by observing intracellular pin trafficking.
in the four arabidopsis long-looped pins  investigated in this and previous studies, the m <dig> motif appears to be generally necessary for proper intracellular trafficking of these long pins.
among arabidopsis pin members, pin <dig> is most closely related to pin <dig> , and their hl amino acid sequences share 74% identity.
previous studies have reported that both pin <dig> and pin <dig> proteins are expressed in the same tissues of the arabidopsis root with the same polarity, such as basally polar in stele cells  <cit>  and apolar in columella cells  <cit> .
these observations suggest that the intracellular behaviors of both pin <dig> and pin <dig> could be regulated by similar intracellular trafficking machinery.
this led us to hypothesize that the function of the m <dig> motif in pin7-hl would be comparable to that in pin3-hl for the regulation of its trafficking.
our previous study showed that phosphorylation-defective m3-pin <dig> was completely internally localized  in the root hair cell  <cit> .
however, the m3-pin <dig> proteins in this study were partly internally localized, and a considerable amount of mutant pin <dig> remained in the pm .
in accordance with its partial pm localization, the mutant pin <dig> proteins showed a different response to bfa from that of m3-pin <dig> in the root hair cell; namely, no overlap of mutant pin <dig>  <cit>  but partial overlap of mutant pin <dig> proteins with the bfa compartments .
the m <dig> motifs of the three long pin subgroups  show distinctive structures .
compared with pin <dig> and pin <dig>  the pin <dig>   <dig>  and  <dig> cluster members have a gap of 6– <dig> amino acid residues between s <dig> and s <dig>  even among pin <dig>   <dig>  and  <dig> cluster members, although the residues between s <dig> and s <dig> are highly conserved, those between s <dig> and t <dig> show considerable sequence divergence .
furthermore, although the expression patterns and subcellular polarities of pin <dig> and pin <dig> are mostly alike, some minor differences in their subcellular polarities have been found.
for example, in the root pericycle cell, pin <dig> is localized to the inner lateral and basal sides  <cit> , whereas pin <dig> shows no polarity in the same cell type  <cit> .
the partially different trafficking behaviors between m3-pin <dig> and m3-pin <dig> in the root hair cell could be due to the minor divergence in the m <dig> motif.
pin <dig> undergoes active degradation under normal growth conditions or in response to environmental stimuli such as gravi-stimulation or dark conditions, and its lytic vacuolar targeting and degradation are associated with ubiquitylation/proteasome activity  <cit> .
these findings imply that diverse molecular cues in pin <dig> are involved in the regulation of its degradation responses.
these molecular cues might be located in the pin2-hl, and this study suggests that the m <dig> motif could be one of them.
several lines of evidence from our results support the idea that the m <dig> motif of pin <dig> is involved in pin <dig> degradation, in particular, by modulating the rate of vacuolar lytic trafficking at least in the root hair cell.
first, 3 m <dig> or m <dig> mutation of pin <dig> decreased cellular pin <dig> proteins to an almost undetectable level .
second, the mutation effect on the pin <dig> protein level seems to be due to degradation but not transcription or translation of pin <dig>  because the transcription of mutant pin <dig> genes was normal  and the wortmannin-restored protein levels of mutant pin <dig> were similar to those of wild-type pin <dig> .
third, 3 m <dig> or m <dig> mutant pin <dig> proteins were restored in the root hair cell by wortmannin , but not by auxin .
because similar protein levels were observed between wild-type and mutant pin <dig> upon wortmannin treatment but mutant pin <dig> proteins were undetectable without wortmannin , it is likely that mutations in the m <dig> phosphorylation sites facilitated the trafficking of mutant pin <dig> proteins to the vacuolar lytic pathway.
on the other hand, the result with mg <dig> is intriguing in that it was not able to inhibit the lytic vacuolar pathway of m3-  pin <dig>  this proteasome inhibitor was found to inhibit the internalization or vacuolar targeting of pin <dig> from the pm  <cit> , and ubiquitylation of pin <dig> is implicated in this process  <cit> .
so far, it remains uncertain how proteasome-mediated degradation contributes to pin <dig> trafficking to the lytic vacuole and whether pin <dig> ubiquitylation is directly linked to proteasome-mediated degradation.
proteasomes are localized in the nucleus and cytoplasm  <cit> , and vacuole-mediated protein degradation is distinct from proteasome-mediated protein degradation  <cit> .
in this context, we can propose two possibilities.
first, ubiquitylation of pin <dig> might act as a signal for vacuolar targeting but not for proteasome-mediated cytoplasmic degradation.
in this case, there could be some cytoplasmic protein factors that are proteasome-sensitive and engaged in the vacuolar trafficking of ubiquitylated pin <dig>  second, cytoplasmic proteasomes and vacuolar proteases might both work together for pin <dig> degradation in a similar way to the degradation of the a-factor transporter  in yeast  <cit> .
the first possibility is more feasible because the inhibitory effect of mg <dig> is more obvious in pin <dig> endocytosis and vacuolar trafficking  <cit> .
the inability of mg <dig> to block the vacuolar trafficking of m3- -pin <dig> in this study prompts us to speculate that phosphorylation of the m <dig>  motif could be necessary for normal vacuolar trafficking of pin <dig>  which is regulated coordinately with other factors such as ubiquitylation of the pin2-hl and proteasome-sensitive trafficking components.
impairment of m <dig>  phosphorylation might disrupt the regulated vacuolar trafficking and redirect pin <dig> trafficking directly to the vacuole.
overall, our results suggest that the m <dig> motif in pin <dig> may have specialized to modulate protein degradation by lytic vacuolar trafficking.
the aforementioned fact that pin <dig> protein degradation is frequently observed under various conditions may reflect that pin <dig> is more susceptible to the protein trafficking machinery for degradation.
the different behavior of m3-  pin <dig> from that of other m3-pins leads us to hypothesize that the specialized function of the pin <dig> m <dig> motif could be due to its specific structure.
the m <dig> motif of pin <dig> not only has additional amino acid residues between s <dig> and s <dig>  but also includes additional serine residues in the m <dig> motif other than the five s/t residues conserved in the long pin m <dig> motif .
furthermore, s <dig> of the arabidopsis pin <dig> m <dig> motif is replaced by alanine.
it is conceivable that these unique features of pin <dig> m <dig> might drive pin <dig> trafficking more frequently to the vacuolar lytic pathway.
including the m <dig> motif, at least three phosphorylation motifs have been characterized from long pin hl domains  <cit> .
the serine residue in the ‘tprxs’  motif of pin1- and pin2-hls has been shown to be phosphorylated and function in pin polarity  <cit> .
long pin hls have three repeats of tprxs, mutations of which additively affect developmental phenotypes  <cit> .
another motif, ‘ser337/thr340’, is required for the proper polarity and biological function of pin <dig>  <cit> .
the m <dig> motif consists of 3 m <dig> and 2 m <dig> regions, with the 2 m <dig> region corresponding to the first tprxs motif .
our previous study showed that mutation of 3 m <dig>  alone in pin <dig> was sufficient to cause phenotypic effects such as alteration of its pm targeting, polarity, and biological function  <cit> .
conversely, 2 m <dig> mutation alone did not have any detectable effect on such phenotypes  <cit> .
furthermore, 3 m <dig> mutation affected pid- or wag1-mediated phosphorylation of pin3-hl whereas 2 m <dig> mutation did not  <cit> .
our previous results with 2 m <dig> mutant pin <dig> were similar to results with pin <dig> mutated at the first tprxs  <cit> , but different from results with pin <dig>  where the mutation of s in the first tprxs significantly decreased pid- or wag-mediated phosphorylation of pin <dig> and affected developmental phenotypes  <cit> .
these results imply that the same tprxs motif from different pins may have different affinity or specificity to different kinases  <cit> .
although mutations of 3 m <dig> and 2 m <dig> together had a much greater effect than 3 m <dig> mutation alone, the 3 m <dig> mutation alone had a considerable effect on the trafficking behavior and activity of long pins .
these data suggest that the 3 m <dig> motif provides long pins with a partly independent phosphorylation code for the modulation of their subcellular trafficking.
however, it should be noted that our current results were based on the root hair model system.
studying individual pins in the cells in which they are most expressed should help identify the role of the 3 m <dig> motif in native pin trafficking and plant development.
previous studies and this study collectively suggest multiple phosphorylation sites in long pin-hls, divergent functions of conserved phosphorylation sites among different pins, and differential targeting by different agc kinases.
in addition, considering the different cell type-specific expression patterns among different pins and kinases, various combinations of the phosphorylation code on the pin-hl would modulate the cell type- and molecule-specific trafficking behavior of long pins.
CONCLUSIONS
our results demonstrate that the m <dig> motif of long pins has been functionally conserved for intracellular pin trafficking.
moreover, divergence in the m <dig> structure among long pins has likely lead to divergence in its specific role in pin trafficking, so that the mutation of m <dig> caused complete  or partial  failure of pm targeting and facilitation of vacuolar lytic trafficking .
the dynamicity of subcellular pin polarity is important to modulate local auxin gradient formation and ultimately diverse developmental processes, which are subject to perpetual changes in environmental and developmental stimuli.
phosphorylation of the pin-hl domain provides pins the ability to change their subcellular trafficking behavior.
furthermore, multiple phosphorylation sites in the pin-hl domain and their divergence in specific structure may generate diverse phosphorylation codes to drive dynamic pin trafficking depending on internal and external stimuli.

 auxin plays important roles in plant growth and development. directional cell-to-cell transport and the formation of concentration gradients of auxin are pivotal for its biological function. diffusive cellular auxin efflux is not effective because of ionization of intracellular auxin, so asymmetrically localized auxin efflux carriers in the plasma membrane play a critical role in polar auxin transport. pin-formed auxin efflux carrier proteins show a distinctive asymmetrical subcellular distribution, which is dynamically regulated by environmental and developmental cues, and their mutations cause defects in auxin gradient formation and plant growth and development. the arabidopsis pin family consists of eight members, among which six pins have a long central hydrophilic loop connecting the five transmembrane helices on each end. these ‘long’ pins mainly localize to the pm. conversely, the other two pins have a very short hl and localize to the internal compartment or to both the internal compartment and the pm. long pins show diverse polar localization in the pm of diverse cell types. although de novo synthesized long pins symmetrically localize to the pm, subsequent cycles of endocytosis and directional recycling processes determine and maintain specific pin polarity. the endocytosis/recycling process of long pins is coordinated and regulated by environmental and cellular signals. protein phosphorylation and dephosphorylation have been implicated in auxin transport and pin trafficking. there is no direct evidence of a role for the tm domain in auxin transport by pins, but several lines of evidence support the idea that the hl acts in regulation of intracellular trafficking of pin proteins. the hl of long pins contains several phosphorylation motifs that are targeted by some kinases. protein kinase inhibitors suppressed cellular auxin transport, and interfered with the trafficking of pin proteins to the pm. mutation or overexpression of pinoid changed auxin transport activity and led to phenotypes associated with auxin transport defects. it has been proposed that changes in phosphorylation status by the antagonistic activities between pid and the protein phosphatase pp2a modulate the subcellular polarity of pins. accordingly, it has been demonstrated that the pin-hl can be phosphorylated by pid and related kinases in vitro and in a protoplast phosphorylation assay, and that the phosphorylation sites of the pin-hl are required for pins’ polarity and biological functions. recently, we reported a phosphorylatable motif, named m in the pin3-hl that includes five s/t residues and is necessary for the polarity and intracellular trafficking of pin. root hair-specific overexpression of wild-type pin greatly inhibited root hair growth by exporting auxin from the root hair cell, whereas overexpression of a phosphorylation-defective m mutant pin failed to inhibit root hair growth. to restore root hair growth, at least three phosphorylation-defective mutations among the five s/t residues were required. the pm localization of pin in root hair cells was disrupted and internalized by the phosphorylation-defective mutations because of the defect in pm targeting. the effect of m phosphorylation-defective mutation on the subcellular localization of pin was also observed in its native expression domain, root pericycle cells, resulting in aberrant localization to the outer lateral pm and defects in gravitropism. the m motif sequence is conserved among all long pins. in this study, using the root hair assay system for pin activity, we investigated whether this motif has been functionally conserved among long pins.  to examine the functional conservation of the m motif among long pins, we chose pin and two other well-studied long pins, pin and pin the five phosphorylatable s/t residues in the m motif are strictly conserved with very few exceptions among long pins including pin and. most of the m s/t residues from pin and are predicted to be phosphorylatable by netphos, as are the pin residues. pin shows a unique feature in that its m motif lacks one putative phosphorylation serine that is replaced by alanine. while the m phosphorylation sites are conserved, there is diversity in the sequence between the phosphorylation sites. these features of m in long pins indicate both conservation and probably specific divergence in function. the m motif is required for the auxin efflux activity of pin pin and pin in the arabidopsis root hair cell in a previous study, we divided the m s/t sites into two sub-motifs, 3 m comprising the first three s residues and 2 m comprising the last two t/s residues. mutation of the 3 m1 s residues caused defects in the phosphorylation, trafficking, efflux activity, and biological functions of pin but 2 m mutation did not. to assess the function of the m motif in the auxin efflux activity of pin pin and pin we generated phosphorylation-defective m and 3 m mutant pin:gfp fusion constructs, which were root-hair specifically expressed under the expansin a promoter in arabidopsis plants. as previously reported, this root hair system was adopted to indirectly estimate the pins’ auxin efflux activity because root hair growth depends on auxin levels in the root hair cell. consistent with our previous report, root hair-specific overexpression of wild-type pin pin or pin greatly inhibited the root hair growth of 4-d-old seedlings by depleting auxin levels in the root hair cell. on the other hand, this pin-mediated root hair inhibition was significantly suppressed by 3 m or m mutation, though m mutation consistently showed a greater restoration of root hair growth than did 3 m mutation, similarly to previous results with pin. among the three pins, the 3 m and m mutants of pin showed the greatest restoration of root hair growth. these results were reproducible with independent transgenic lines for each pin. in the case of pin 3 m and m mutations suppressed the auxin efflux activity, and thus root hair inhibition, of pin by interrupting its trafficking to the pm. to test whether this was the same for pin pin and pin we examined the subcellular localization of the mutant forms of these pins. the m motif is required for pm targeting of pin and pin in the root hair cell to determine whether the mutant pins were pm or internally localized, gfp-tagged pins were first co-visualized with the endocytic tracer fm4- in the root hair cell. wild-type pin and pin overlapped with the fm4- signal at the pm, whereas the 3 m1- and m3-mutant forms of pin and pin showed considerable internal localization, though they were partly localized in the pm as well. this internal localization pattern of mutated pin and pin was consistently observed in independent transgenic lines. observation of two representative transgenic lines of 3 m1- and m3-pins showed that pin internalization occurred in ~50% of the root hair cells, whereas, in the case of pin internalization occurred in ~30% of 3 m and ~55% of m mutant transgenic lines under similar conditions. these data support the idea that the probable phosphorylation-defective mutations in the 3 m or m motifs significantly impaired the capability of pin and pin to target to the pm and thus resulted in suppression of pin-mediated root hair inhibition. the exocytosis inhibitor brefeldin a causes internal accumulation of pin proteins into so-called ‘bfa compartments’, which are indicative of pm-localization of pins. to clarify the subcellular localization of mutant pin and pin proteins, the transgenic seedlings were co-treated with the protein synthesis inhibitor cycloheximide and bfa. signals of wild-type pin and pin proteins completely overlapped with those of fm4- in the bfa compartments, whereas 3 m or m mutant pin and pin proteins only partly co-localized with the fm4-64-stained bfa compartments. the lack of internal mutant pin signal and bfa compartment overlap further supports the idea that the ability of pin and pin to target to the pm was partially disrupted by the 3 m and m phosphorylation-defective mutations. m mutations lead to rapid degradation of pin through the vacuolar lytic pathway in the root hair cell unlike phosphorylation-defective pin and pin the mutant pin proteins showed completely different trafficking behavior. while wild-type pin was clearly localized to the pm, phosphorylation-defective 3 m1- or m3-pin proteins were barely detectable in root hair cells; this was consistent in all five independent transgenic lines observed for each mutant construct. this result was in accordance with the results from the root-hair-inhibition assay, where 3 m or m mutant pin2s showed much weaker inhibitory effects on root hair growth than did mutant pin or pin. the very low expression of these mutant pin proteins in the cell could be due to either translational failure or degradation of the mutant proteins because the mutant transgenes seemed to be normally transcribed, as shown in the rt-pcr analysis. because there were some residual inhibitory effects on root hair growth as mentioned above, it is conceivable that a certain portion of mutant pin proteins might stay transiently in the pm, resulting in weak auxin-exporting activity, and then undergo endocytosis and degradation. to test this idea and to restore the mutant pin protein in the cell, we applied auxin to inhibit clathrin-dependent pin endocytosis from the pm, mg to inhibit ubiquitylation/proteasome-mediated pin internalization and degradation, and wortmannin to inhibit the vacuolar lytic pathway. naa did not have much effect on the pm-localization of wild-type pin or restored 3 m1- or m3-pin proteins in the root hair cell. mg caused partial internal accumulation of wild-type pin proteins, but failed to recover the mutant pin signals in the root hair cell. wortmannin, an inhibitor of phosphatidylinositol-3-kinase, inhibits the trafficking of pin proteins as well as fm4- to the vacuole. wortmannin treatment restored the gfp signals of 3 m and m mutant pin2:gfp proteins in the root hair cell. when five independent lines for each of two mutant pin constructs were observed, the intensities of wortmannin-restored mutant pin signals were similar to those of wild-type pin, implying that the translational efficiency of the wild-type and mutant pin2s was similar, but that the 3 m or m phosphor-defective pin proteins tended to enter the lytic pathway more than the wild-type protein. discussion in this study, we investigated whether the m motif, a phosphorylation motif originally found in the pin3-hl that regulates pin3’s polarity and intracellular trafficking, has been functionally conserved in other long-looped pins using the root hair assay system to estimate pin activity and by observing intracellular pin trafficking. in the four arabidopsis long-looped pins investigated in this and previous studies, the m motif appears to be generally necessary for proper intracellular trafficking of these long pins. among arabidopsis pin members, pin is most closely related to pin, and their hl amino acid sequences share 74% identity. previous studies have reported that both pin and pin proteins are expressed in the same tissues of the arabidopsis root with the same polarity, such as basally polar in stele cells and apolar in columella cells. these observations suggest that the intracellular behaviors of both pin and pin could be regulated by similar intracellular trafficking machinery. this led us to hypothesize that the function of the m motif in pin7-hl would be comparable to that in pin3-hl for the regulation of its trafficking. our previous study showed that phosphorylation-defective m3-pin was completely internally localized in the root hair cell. however, the m3-pin proteins in this study were partly internally localized, and a considerable amount of mutant pin remained in the pm. in accordance with its partial pm localization, the mutant pin proteins showed a different response to bfa from that of m3-pin in the root hair cell; namely, no overlap of mutant pin but partial overlap of mutant pin proteins with the bfa compartments. the m motifs of the three long pin subgroups show distinctive structures. compared with pin and pin the pin and cluster members have a gap of 6– amino acid residues between s and s even among pin and cluster members, although the residues between s and s are highly conserved, those between s and t show considerable sequence divergence. furthermore, although the expression patterns and subcellular polarities of pin and pin are mostly alike, some minor differences in their subcellular polarities have been found. for example, in the root pericycle cell, pin is localized to the inner lateral and basal sides, whereas pin shows no polarity in the same cell type. the partially different trafficking behaviors between m3-pin and m3-pin in the root hair cell could be due to the minor divergence in the m motif. pin undergoes active degradation under normal growth conditions or in response to environmental stimuli such as gravi-stimulation or dark conditions, and its lytic vacuolar targeting and degradation are associated with ubiquitylation/proteasome activity. these findings imply that diverse molecular cues in pin are involved in the regulation of its degradation responses. these molecular cues might be located in the pin2-hl, and this study suggests that the m motif could be one of them. several lines of evidence from our results support the idea that the m motif of pin is involved in pin degradation, in particular, by modulating the rate of vacuolar lytic trafficking at least in the root hair cell. first, 3 m or m mutation of pin decreased cellular pin proteins to an almost undetectable level. second, the mutation effect on the pin protein level seems to be due to degradation but not transcription or translation of pin because the transcription of mutant pin genes was normal and the wortmannin-restored protein levels of mutant pin were similar to those of wild-type pin. third, 3 m or m mutant pin proteins were restored in the root hair cell by wortmannin, but not by auxin. because similar protein levels were observed between wild-type and mutant pin upon wortmannin treatment but mutant pin proteins were undetectable without wortmannin, it is likely that mutations in the m phosphorylation sites facilitated the trafficking of mutant pin proteins to the vacuolar lytic pathway. on the other hand, the result with mg is intriguing in that it was not able to inhibit the lytic vacuolar pathway of m3- pin this proteasome inhibitor was found to inhibit the internalization or vacuolar targeting of pin from the pm, and ubiquitylation of pin is implicated in this process. so far, it remains uncertain how proteasome-mediated degradation contributes to pin trafficking to the lytic vacuole and whether pin ubiquitylation is directly linked to proteasome-mediated degradation. proteasomes are localized in the nucleus and cytoplasm, and vacuole-mediated protein degradation is distinct from proteasome-mediated protein degradation. in this context, we can propose two possibilities. first, ubiquitylation of pin might act as a signal for vacuolar targeting but not for proteasome-mediated cytoplasmic degradation. in this case, there could be some cytoplasmic protein factors that are proteasome-sensitive and engaged in the vacuolar trafficking of ubiquitylated pin second, cytoplasmic proteasomes and vacuolar proteases might both work together for pin degradation in a similar way to the degradation of the a-factor transporter in yeast. the first possibility is more feasible because the inhibitory effect of mg is more obvious in pin endocytosis and vacuolar trafficking. the inability of mg to block the vacuolar trafficking of m3- -pin in this study prompts us to speculate that phosphorylation of the m motif could be necessary for normal vacuolar trafficking of pin which is regulated coordinately with other factors such as ubiquitylation of the pin2-hl and proteasome-sensitive trafficking components. impairment of m phosphorylation might disrupt the regulated vacuolar trafficking and redirect pin trafficking directly to the vacuole. overall, our results suggest that the m motif in pin may have specialized to modulate protein degradation by lytic vacuolar trafficking. the aforementioned fact that pin protein degradation is frequently observed under various conditions may reflect that pin is more susceptible to the protein trafficking machinery for degradation. the different behavior of m3- pin from that of other m3-pins leads us to hypothesize that the specialized function of the pin m motif could be due to its specific structure. the m motif of pin not only has additional amino acid residues between s and s but also includes additional serine residues in the m motif other than the five s/t residues conserved in the long pin m motif. furthermore, s of the arabidopsis pin m motif is replaced by alanine. it is conceivable that these unique features of pin m might drive pin trafficking more frequently to the vacuolar lytic pathway. including the m motif, at least three phosphorylation motifs have been characterized from long pin hl domains. the serine residue in the ‘tprxs’ motif of pin1- and pin2-hls has been shown to be phosphorylated and function in pin polarity. long pin hls have three repeats of tprxs, mutations of which additively affect developmental phenotypes. another motif, ‘ser337/thr340’, is required for the proper polarity and biological function of pin. the m motif consists of 3 m and 2 m regions, with the 2 m region corresponding to the first tprxs motif. our previous study showed that mutation of 3 m alone in pin was sufficient to cause phenotypic effects such as alteration of its pm targeting, polarity, and biological function. conversely, 2 m mutation alone did not have any detectable effect on such phenotypes. furthermore, 3 m mutation affected pid- or wag1-mediated phosphorylation of pin3-hl whereas 2 m mutation did not. our previous results with 2 m mutant pin were similar to results with pin mutated at the first tprxs, but different from results with pin where the mutation of s in the first tprxs significantly decreased pid- or wag-mediated phosphorylation of pin and affected developmental phenotypes. these results imply that the same tprxs motif from different pins may have different affinity or specificity to different kinases. although mutations of 3 m and 2 m together had a much greater effect than 3 m mutation alone, the 3 m mutation alone had a considerable effect on the trafficking behavior and activity of long pins. these data suggest that the 3 m motif provides long pins with a partly independent phosphorylation code for the modulation of their subcellular trafficking. however, it should be noted that our current results were based on the root hair model system. studying individual pins in the cells in which they are most expressed should help identify the role of the 3 m motif in native pin trafficking and plant development. previous studies and this study collectively suggest multiple phosphorylation sites in long pin-hls, divergent functions of conserved phosphorylation sites among different pins, and differential targeting by different agc kinases. in addition, considering the different cell type-specific expression patterns among different pins and kinases, various combinations of the phosphorylation code on the pin-hl would modulate the cell type- and molecule-specific trafficking behavior of long pins.  our results demonstrate that the m motif of long pins has been functionally conserved for intracellular pin trafficking. moreover, divergence in the m structure among long pins has likely lead to divergence in its specific role in pin trafficking, so that the mutation of m caused complete or partial failure of pm targeting and facilitation of vacuolar lytic trafficking. the dynamicity of subcellular pin polarity is important to modulate local auxin gradient formation and ultimately diverse developmental processes, which are subject to perpetual changes in environmental and developmental stimuli. phosphorylation of the pin-hl domain provides pins the ability to change their subcellular trafficking behavior. furthermore, multiple phosphorylation sites in the pin-hl domain and their divergence in specific structure may generate diverse phosphorylation codes to drive dynamic pin trafficking depending on internal and external stimuli. 
root hair-specific overexpression of wild-type pin or greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of m phosphorylation-defective pin mutants failed to inhibit root hair growth. consistent with this root hair phenotype, the pm localization of m phosphorylation-defective pin and pin was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. however, the mutant pin protein levels were restored by wortmannin treatment almost to the wild-type pin level, indicating that the m motif of pin unlike that of other pins, is implicated in pin trafficking to the vacuolar lytic pathway. these results suggest that the m phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-hl pins, but its specific function in trafficking has diverged among pin members. pin-formed efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane. recently, we reported that a phosphorylatable motif in the pin3-hl is necessary for the polarity, intracellular trafficking, and biological functions of pin in this study, using the root hair system for pin activity assay, we investigated whether this motif has been functionally conserved among long-hl pins.
 pin-formed efflux carriers contribute to polar auxin transport and plant development by exhibiting dynamic and diverse asymmetrical localization patterns in the plasma membrane. phosphorylation of the central hydrophilic loop of pins has been implicated in the regulation of pin trafficking. recently, we reported that a phosphorylatable motif in the pin3-hl is necessary for the polarity, intracellular trafficking, and biological functions of pin in this study, using the root hair system for pin activity assay, we investigated whether this motif has been functionally conserved among long-hl pins.  root hair-specific overexpression of wild-type pin or greatly inhibited root hair growth by depleting auxin levels in the root hair cell, whereas overexpression of m phosphorylation-defective pin mutants failed to inhibit root hair growth. consistent with this root hair phenotype, the pm localization of m phosphorylation-defective pin and pin was partially disrupted, resulting in less auxin efflux and restoration of root hair growth. partial formation of brefeldin a-compartments in these phosphorylation-mutant pin lines also suggested that their pm targeting was partially disrupted. on the other hand, compared with the pin and pin mutant proteins, m3-phosphorylation-defective pin proteins were almost undetectable. however, the mutant pin protein levels were restored by wortmannin treatment almost to the wild-type pin level, indicating that the m motif of pin unlike that of other pins, is implicated in pin trafficking to the vacuolar lytic pathway.  these results suggest that the m phosphorylation motif has been functionally conserved to modulate the intracellular trafficking of long-hl pins, but its specific function in trafficking has diverged among pin members. auxinauxin transporthydrophilic loop phosphorylationpin-formed protein traffickingroot hair 