The results obtained in this work demonstrate that tomato C22DES is an ER-resident protein. The 3D modeling of tomato C22DES using the crystal structure of the phylogenetically related lanosterol 14α-demethylase (CYP51), an enzyme also involved in sterol metabolism thought to be one of the most ancient and conserved P450s across the kingdoms, predicted the presence of an N-terminal hydrophobic transmembrane domain and two short sequences (MCR1 and MCR2) located in the globular domain which also interact with the ER membrane (Fig. 4). Subcellular localization studies using C22DES derivatives fused to GFP indicated that TMH is sufficient for the targeting and retention of C22DES in the ER (Figs. 5 and 6D). These studies also revealed that the globular domain can interact and be retained in the ER membrane in the absence of TMH (Fig. 6). The sequence conservation of MCR1 and MCR2 among plant C22DES (Figs. 4C and D) supports a role for these sequences in the interaction with the ER membrane.
The observation that the globular domain of tomato C22DES was enzymatically inactive despite behaving as an integral membrane protein (Fig. 7) revealed that TMH was also required for enzyme activity. This result was somehow unexpected considering the recent work of Gnanasekaran et al. (2015) showing that the N-terminal region of CYP720B4, a plant cytochrome P450 involved in isopimeric acid biosynthesis, was not essential for the activity of this enzyme when expressed in N. benthamiana leaves. However, the functional role of the N-terminal transmembrane domain of CYPs remains a controversial issue. Thus, while some recombinant CYPs lacking the N-terminal anchor region have been reported to be active in vitro, no activity could be detected in in vivo assays (34, 45, 46). The differential behavior of the N-terminal region of CYPs may be related, at least in part, with the nature and subcellular availability of their substrates. This would explain why some CYPs show activity under in vitro test conditions in which the substrate is fully available, but not under in vivo assay conditions where the availability of substrate may be a limiting factor. Furthermore, it has been proposed that the N-terminal membrane domain of some CYPs participates in the correct positioning of the globular domain with respect to the membrane during catalysis. Thus, it has been reported that the transient tilting of the globular domain is an essential requirement to allow the interaction of CYPs with their substrates when located within the hydrophobic core of the ER membrane (47, 48). In the particular case of C22DES, it can be speculated that it could not interact with its substrate (β-sitosterol) unless TMH provides the right anchoring of the globular domain to allow its tilting within the ER membrane during catalysis.
Despite the essential role of TMH in tomato C22DES activity, it was surprising to find that both the length and the amino acid sequence of the N-terminal region of plant C22DES was poorly conserved (Fig. 8). However, a detailed inspection of these sequences revealed two conserved features that could be relevant for enzyme activity: i) the presence of a cholesterol recognition/interaction amino-acid consensus (CRAC) motif, and ii) an enrichment of serine and threonine residues in the N-terminal half of TMH. Cholesterol-binding domains have been the focus of many studies involving computational methods to explore the transmembrane regions of animal proteins for which there is good evidence of their interaction with cholesterol (41, 42). The first motif to be identified was termed cholesterol recognition/interaction amino-acid consensus (CRAC) and fulfills the consensus (L/V)-X1-5-(Y)-X1-5-(R/K), (where X is any amino acid) (41, 42, 49). Another cholesterol-binding motif named CARC corresponds to the mirror version of the CRAC motif with the consensus sequence (K/R)-X1-5-(Y/F)-X1-5-(L/V) (42, 49). Despite the CRAC motif was initially identified and characterized in animal proteins (49–52), its cholesterol-binding function has also been demonstrated in plants (53). Interestingly, a CRAC motif able to interact with β-sitosterol has recently been described in the type 1 cholecystokinin receptor (54). Thus, it is likely the CRAC1 motif present in the N-terminal region of plant C22DES (CRAC1) may contribute to the interaction of the enzyme with the β-sitosterol present in the ER membrane. Another feature reported in cholesterol-binding regions is the presence of serine and threonine residues. Furthermore, the structural analysis of several cholesterol-binding proteins has led to propose that the hydrophilic side-chain of these amino acids may interact with the C3-hydroxyl group of the cholesterol molecule (55). Thus, it is likely that the serine and threonine residues present in the N-terminal half of TMH could enhance the interaction of C22DES with β-sitosterol in the ER, facilitating in this way its interaction with the CRAC1 motif.
The localization of C22DES in the ER was not unexpected considering that other enzymes involved in sterol biosynthesis, such as SMT1, CPI1, HYD1, and DWF/DIM (5, 56–58) as well as cytochrome P450 reductase, the physiological redox partner of cytochrome P450s, also localize in this cellular compartment (59). However, the localization of C22DES in the ER raises the question about how this enzyme can act on the major cellular pool of b-sitosterol present in the PM. It is likely that this process may involve the capacity of the ER to physically interact with the PM at structures known as ER-PM contact sites in which both membranes are in close contact (60–62). The possibility that enzymes located in one membrane may act on substrates present in a different cell membrane, the so-called in trans activity, is not unprecedented (63–66). Interestingly, specific ER-PM contact sites involved in the regulation of lipid-homeostasis, including phospholipids and sterols, have recently been reported (67). Furthermore, enzymes involved in lipid biosynthesis have been localized at ER-PM contact sites and shown to contribute to their formation (66, 68). However, and to the best of our knowledge, the involvement of ER-PM contact sites in sterol or lipid homeostasis has not yet been reported in plants, although it has been suggested that the PM-localized tomato Acyl-CoA:sterol acyltransferase (SlASAT1) may act in trans on its substrate cycloartenol in the ER lipid bilayer at ER–PM contact sites to produce cycloartenyl esters (69). The existence of several conserved CRAC/CARC motifs in the globular domain of plant C22DES suggests that they could have a role in the interaction of the enzyme with the β-sitosterol present in the PM. In this respect, the localization of CARC2, CARC3 and CRAC3/CARC4 in amphyphathic α-helices may be relevant considering that this type of structures has been described to serve not only as membrane interaction domains but also as lipid binding sites (70). The study of the contribution of these CARC and CRAC motifs in the activity of C22DES on the β-sitosterol present in the PM represents an interesting issue in further studies dealing with the functional characterization of this enzyme and the regulation of plant sterol metabolism.