The PIN-FORMED (PIN) protein family of auxin transporters
© BioMed Central Ltd 2009
Published: 29 December 2009
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© BioMed Central Ltd 2009
Published: 29 December 2009
The PIN-FORMED (PIN) proteins are secondary transporters acting in the efflux of the plant signal molecule auxin from cells. They are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. PIN genes are found exclusively in the genomes of multicellular plants and play an important role in regulating asymmetric auxin distribution in multiple developmental processes, including embryogenesis, organogenesis, tissue differentiation and tropic responses. All PIN proteins have a similar structure with amino- and carboxy-terminal hydrophobic, membrane-spanning domains separated by a central hydrophilic domain. The structure of the hydrophobic domains is well conserved. The hydrophilic domain is more divergent and it determines eight groups within the protein family. The activity of PIN proteins is regulated at multiple levels, including transcription, protein stability, subcellular localization and transport activity. Different endogenous and environmental signals can modulate PIN activity and thus modulate auxin-distribution-dependent development. A large group of PIN proteins, including the most ancient members known from mosses, localize to the endoplasmic reticulum and they regulate the subcellular compartmentalization of auxin and thus auxin metabolism. Further work is needed to establish the physiological importance of this unexpected mode of auxin homeostasis regulation. Furthermore, the evolution of PIN-based transport, PIN protein structure and more detailed biochemical characterization of the transport function are important topics for further studies.
The first PIN family members identified and associated with auxin transport were described in the model plant Arabidopsis thaliana. The significance and function of AtPIN1 was discovered through the phenotype generated by the loss-of-function mutation in the gene: mutant plants fail to develop floral organs properly and generate naked, pin-like inflorescences, which gave the name PIN-FORMED (PIN) to the family [5, 6]. At the same time, several groups identified the homologous protein AtPIN2 under different names on the basis of a strong root agravitropic phenotype of the loss-of-function mutant. Independently identified mutant alleles of PIN2 were pin2, ethylene insensitive root1 (eir1), agravitropic1 (agr1), and wavy6 (wav6) [7–10]. Altogether, Arabidopsis has eight annotated PIN genes, of which six have been functionally characterized up to now: PIN1 , PIN2 [7–10], PIN3 , PIN4 , PIN5 , and PIN7 . PIN6 and PIN8 are still awaiting characterization.
With the possible exception of the PIN6-related proteins, the general function of all long PINs from seed plants is to transport auxin out of the cell. The groups differ in the regulation of their expression, localization and activity rather than in the auxin-transport function itself. It has been shown, for example, that AtPIN1 and AtPIN2, which are distinct representatives of the long PINs, can functionally replace each other in planta when expressed in the same cells and localized at the same side of the cell [16, 19].
The second major PIN gene subfamily encodes proteins with the central hydrophilic loop virtually absent ('short' PINs) and comprises AtPIN5 and AtPIN8. Sequence diversification within the subfamily of short PINs tends to be higher than between the long PINs. From this subfamily, only AtPIN5 has been characterized so far , and reveals a striking difference from the canonical long PINs in its subcellular localization and thus in its physiological function (see below). The short PINs appear to localize to a large extent to the endoplasmic reticulum, and although they presumably act as auxin transporters, they do not directly facilitate auxin transport between cells but mediate intracellular auxin compartmentalization and homeostasis .
The precise origin of PIN proteins in the evolutionary history of plants is not known. The basal split of the Viridiplantae - that is, the separation of the Streptophyta (the clade containing land plants (Embryophyta) and some green algae) from the Chlorophyta (representing the majority of green algae) - probably occurred some 725-1,200 million years ago  (Figure 1). All green algae with genomes sequenced so far (Chlamydomonas, Ostreococcus and Micromonas) belong to the clade Chlorophyta and none of these organisms contains a PIN gene. On the other hand, sequence data from the most primitive land plants available - the moss Physcomitrella patens and the club moss Selaginella moellendorffii - have revealed the presence of PIN genes of groups 1 and 2, both belonging to the long PIN subfamily. Nonetheless, to assess the evolutionary origin of PIN proteins more precisely, the genomic data from algae more closely related to land plants (that is, from the Streptophyta) and also from the liverworts, land plants even more ancient than the club mosses, is needed. Interestingly, the P. patens and S. oellendorffii PINs do not cluster with PINs of seed plants or with each other (Figure 3, groups 1 and 2), suggesting separate evolutionary establishment of PIN families in each of the lineages. The only exception is P. patens PpPIND (accession number XP_001765763), which is in the same group as AtPIN6. However, its intron sequences suggest the possibility of horizontal transfer of this gene from monocots .
The predicted structure of canonical long PIN proteins is similar to the structures of secondary transporters - that is, membrane transport proteins that use the electrochemical gradient across the membrane, rather than ATP hydrolysis, to power transport. The PIN proteins have two hydrophobic domains (each with five transmembrane helices) that are separated by a hydrophilic domain with a presumably cytoplasmic orientation. This predicted structure is based only on bioinformatic analyses of the sequen available and has not been verified experimentally. The hydrophobic domains of PIN proteins are highly conserved in sequence, mainly in the transmembrane helices, which tolerate no insertions or deletions; the loops between the transmembrane helices within the hydrophobic domains exhibit much greater variability both in size and sequence. The hydrophilic domains of PIN proteins from the same group (Figure 3) are very similar in sequence, but there is only limited sequence similarity between hydrophilic domains of PINs from different groups.
There is a substantial difference in the sequence variability of the hydrophobic domains between short and long PINs. The hydrophobic domains of long PINs contain positions that have the same amino acid in all available sequences - that is, they are invariant - but not all of these positions are invariant in the short PINs. However, there are no amino-acid positions that are invariant in short PINs but not in the long PINs (Figure 2). This indicates that the positions that are invariant only in long PINs must be crucial for some important function of long PINs that has not been retained in short PINs.
Two motifs important for intracellular trafficking of PIN proteins can be predicted. One comprises two diacidic motifs presumably important for trafficking of proteins from the endoplasmic reticulum that are located in the amino-terminal part of the hydrophilic domain of all long PINs. The other is a tyrosine-based internalization motif present in all PINs that is important for recruitment of the protein into clathrin-dependent vesicles. The importance of these residues for PIN action, however, remains to be demonstrated.
PIN promoter activity can be flexibly regulated, which accounts for a compensatory type of functional redundancy. Several pin knockout mutants in Arabidopsis show ectopic activity of other PIN proteins compensating for the lost PIN activity . This phenomenon seems to account for the high degree of functional redundancy among PIN genes, masking most of the phenotypic manifestations expected to result from single, and some double, PIN gene inactivations [14, 23, 24].
The PIN proteins mediate asymmetric auxin distribution within tissues, and various endogenous and exogenous signals modulate auxin distribution and thus plant development by acting on PIN proteins. PIN protein activity can be regulated at many levels, including regulation of transcription, protein degradation, subcellular trafficking (endocytic recycling and polarized targeting) and transport activity [3, 4, 25]. For many of the Arabidopsis PIN genes, regulation by other hormonal pathways has been demonstrated. Auxin itself upregulates the transcription of many long PINs. In contrast, the 'short' AtPIN5 is downregulated by auxin . Other phytohormones and plant growth regulators also influence the activity of the PIN promoters to various degrees. The effects are organ- or even cell-type-specific and strongly depend on the particular part of the plant examined and growth regulator used (brassinosteroids [26–28], cytokinins [29–31], gibberellins , ethylene , flavonoids [34, 35]). PIN abundance is also regulated at the level of protein stability. Several PIN proteins, mainly AtPIN2, exhibit pronounced auxin-regulated turnover based on PIN trafficking to the vacuole and their degradation there [36–38].
The only genetically characterized member of the short PIN subfamily is AtPIN5. Its auxin-transport function (shown in yeast cells) together with its subcellular localization at the endoplasmic reticulum membrane implies the transport of auxin molecules from the cytosol into the lumen of endoplasmic reticulum. As a result of this translocation, auxin molecules are exposed to metabolic enzymes localized in the endoplasmic reticulum, leading to metabolic changes that decrease the availability of free active auxin molecules in the cytosol. In this way, AtPIN5 contributes to control of intracellular auxin homeostasis .
In contrast to the wealth of data on the developmental roles of PIN proteins, there is only limited knowledge on their structure, their structure-function relationships and the mechanism of transport. Earlier physiological experiments  established that auxin efflux requires a membrane H+ gradient. Moreover, no ATP-binding motifs suggesting ATP-dependent transport have been recognized in PIN protein sequences. These findings, together with PIN topology in the membrane, suggest that the PIN proteins are gradient-driven secondary transporters. In particular physiological situations, they can act cooperatively with the ATP-dependent auxin transporters of the ABCB (ATP-binding cassette B) family [53, 54].
Out of the eight PIN proteins in Arabidopsis, the canonical long PINs are already well characterized and their developmental roles in generating intercellular auxin distribution patterns have been demonstrated . On the other hand, the existence of auxin transport into the endoplasmic reticulum and its role in regulating auxin homeostasis is a novel and unexpected finding and there is still lot of work needed to elucidate the details and physiological importance of this activity. From the evolutionary point of view, it would be interesting to know which function of PINs is the older: the plasma-membrane-based intercellular auxin transport by long PINs or the endoplasmic-reticulum-based control of intracellular auxin homeostasis by short PINs? The most ancient PIN proteins currently known, from mosses, are localized to the endoplasmic reticulum, which suggests that intracellular function is evolutionarily ancestral, but this remains to be experimentally verified. The other obvious open questions relate to experimental information on PIN protein structure and membrane topology. This, as well as more detailed biochemical characterization of PIN-driven auxin transport is still largely lacking.
The authors acknowledge the support for their work from the Ministry of Education of the Czech Republic, project LC06034 (PK, PS, JL, EZ), from the Grant Agency of the ASCR, project KJB600380904 (JL), and IAA601630703 (JF).