Ptide carriers present in S. cerevisiae, i.e. within the mutant
Ptide carriers present in S. cerevisiae, i.e. inside the mutant SphK1 custom synthesis strain opt1 dal5 ptr2 (Fig. 5A) (Hauser et al., 2000; 2001; Cai et al., 2007). However, L-citrulline transport was still inhibited by L-Asp–L-Phe in this triple mutant, indicating interaction from the dipeptide with Gap1 no matter the absence of peptide carrier-mediated transport (Fig. S7A and B). Development on many dipeptides and tripeptides as only nitrogen α9β1 Species supply was impaired in cells deleted for these 3 big peptide carriers. For example, wild-type and gap1 cells could use 1 mM of Leu-Met-NH2 or L-Arg-Gly-Gly [two non-competitive inhibitors of Gap1-dependent Lcitrulline transport (Van Zeebroeck et al., 2009)], indicating that these two peptides do not enter cells through Gap1 (Fig. 5B). However, the strain opt1 dal5 ptr2 could no longer use them as only N source, presumably mainly because of its inability to take them up (Fig. 5B). In contrast, L-Asp-L-Phe couldn’t be employed as only nitrogen supply either by the wild-type or by the gap1 strain indicating that even when it is transported inside the cells it really is not metabolized (Fig. 5A and B). L-Asp–L-Phe was hence a good candidate to test ubiquitination and endocytosis by a non-transported substrate analogue, given that it still inhibits L-citrulline transport inside the opt1 dal5 ptr2 strain (Fig. S7) (Van Zeebroeck et al., 2009). Regardless of its uptake by the peptide carriers, this dipeptide was unable to induce endocytosis of Gap1-GFP, as shown in either wild-type or opt1 dal5 ptr2 strains (Fig. 5C). Hence, its interaction with Gap1 is just not sufficient to trigger Gap1 endocytosis. Even so, when we tested appearance of oligo-ubiquitinated types in cells of the wild-type or the opt1 dal5 ptr2 strain expressing myc-Ubi upon exposure to L-Asp–L-Phe, we clearly detected look and accumulation of di- and triubiquitinated types of Gap1 in both situations (Fig. 5D). Theiraccumulation was much more permanent than within the case of L-citrulline. Quantification revealed a two- to threefold increase, equivalent to the intensity in the transient boost in oligo-ubiquitination observed with L-citrulline. This indicated that though the interaction of L-Asp–L-Phe with Gap1 will not suffice to bring about Gap1 endocytosis it still causes substantial accumulation of oligo-ubiquitinated Gap1. This really is towards the ideal of our knowledge the initial case of a non-transported molecule causing ubiquitination of a transporter (or transceptor). In addition, this outcome confirms that oligo-ubiquitination isn’t adequate per se to trigger endocytosis of a transporter (or transceptor), suggesting that added alterations e.g. in conformation or in posttranslational modification can be needed to initiate endocytosis. An alternative possibility for all the cases where we’ve got observed an apparent lack of endocytosis is that endocytosis is masked by enhanced accumulation of newly synthesized Gap1 arriving at the plasma membrane. To evaluate this possibility we tested plasma membrane localization of Gap1-GFP soon after addition of the compounds that are unable to trigger substantial endocytosis, L-Lys, L-Asp–L-Phe, and D-His, in circumstances in which protein translation is abolished by addition of 50 g ml-1 from the protein synthesis inhibitor, cycloheximide (Fig. S8). To ensure that translation was stopped at the starting of the experiment, the cells have been pre-incubated for 20 min within the presence of cycloheximide. If the stable plasma membrane signal outcomes from accumulation of newly.