Ly of either Yif1A/Yif1p or the Ypt1p/Ypt31p category of Rab GTPases. Pertaining to this hypothesis, RNAi-mediated Yif1A knockdown was previously reported to cause a fragmentation of the Golgi apparatus, consistent with an ER-to-Golgi trafficking defect, but no ER phenotype was reported [13]. To support our conjecture that Yip1A-mediated control of ER whorl formation does not depend on Yif1A, we reproduced the previously published Yif1A knockdown but with an eye towards revealing any potential ER phenotypes. Consistent with a lack of ML240 chemical information requirement for Yif1A in ER structural maintenance, we observed no ER whorls in cells depleted of Yif1A, even though Golgi fragmentation was clearly and frequently observed (Fig. 5).Ala/Leu replacements of transmembrane segments reveal crucial residuesFor dissecting the required regions of the TM domain, we relied on both secondary structure and TM domain prediction algorithms (schematized in Fig. 3B) to make truncations of individual TM helices and loops so as to minimize perturbation of overall membrane topology. Unfortunately, none of the resulting deletion constructs led to stably expressed protein (data not shown). To bypass this issue, we proceeded by replacing segments of predicted TM helices with Ala 3PO residues interspersed with Leu so as to maintain their stability in the membrane [34]; similarly, predicted luminal and cytoplasmic loops were replaced with stretches of Ala. In the case of charged residues, charge reversal mutations were made to maximize the potential effect of the mutation. All of the constructs made were expressed, except for those with substitutions in TM3 and TM4. Curiously, even single amino acid substitutions within TM3 and TM4 led to poorly expressed protein, precluding their analysis. Nonetheless all other TM helices and loops were amenable to substitutions, allowing a dissection of the remainder of the TM domain. Each expressing construct was tested in our knockdown replacement assay and the results are quantified in Fig. 3A and schematized in Fig. 3B. (A full table of mutations and results is included in Table S1). The analysis revealed substantial stretches where residue identity was seemingly not important for function, such as all of TM1 and Loop2; however for others, such as TM2 and Loop1, substitutions of 7?0 amino acid stretches disrupted function. To further refine the required residues, we constructed point mutations within the seemingly required regions. Surprisingly, when variants with individual amino acid substitutions were produced, it became clear that only two point mutations, K146E and V152L, resulted in any loss of function, though the loss was modest (Fig. 4A, D). Given their proximity to one another, we also tested the effect of combining the two substitutions. Notably, the double mutant variant K146E/V152L was completely nonfuncDiscussion The residue identity of surprisingly few amino acids tested are important for the mechanism of Yip1A functionAs shown previously for its yeast counterpart in a viability assay [19], the majority of the cytoplasmic domain of Yip1A was dispensable for its ER structural maintenance function. This dispensable portion has no predicted secondary structure; therefore it is difficult to speculate on its potential role. Yet its persistence through evolution suggests a role, perhaps regulatory, under conditions yet-to-be assessed. In contrast to the cytoplasmic domain, we found that the membrane-spanning domain of Yip1AMutational A.Ly of either Yif1A/Yif1p or the Ypt1p/Ypt31p category of Rab GTPases. Pertaining to this hypothesis, RNAi-mediated Yif1A knockdown was previously reported to cause a fragmentation of the Golgi apparatus, consistent with an ER-to-Golgi trafficking defect, but no ER phenotype was reported [13]. To support our conjecture that Yip1A-mediated control of ER whorl formation does not depend on Yif1A, we reproduced the previously published Yif1A knockdown but with an eye towards revealing any potential ER phenotypes. Consistent with a lack of requirement for Yif1A in ER structural maintenance, we observed no ER whorls in cells depleted of Yif1A, even though Golgi fragmentation was clearly and frequently observed (Fig. 5).Ala/Leu replacements of transmembrane segments reveal crucial residuesFor dissecting the required regions of the TM domain, we relied on both secondary structure and TM domain prediction algorithms (schematized in Fig. 3B) to make truncations of individual TM helices and loops so as to minimize perturbation of overall membrane topology. Unfortunately, none of the resulting deletion constructs led to stably expressed protein (data not shown). To bypass this issue, we proceeded by replacing segments of predicted TM helices with Ala residues interspersed with Leu so as to maintain their stability in the membrane [34]; similarly, predicted luminal and cytoplasmic loops were replaced with stretches of Ala. In the case of charged residues, charge reversal mutations were made to maximize the potential effect of the mutation. All of the constructs made were expressed, except for those with substitutions in TM3 and TM4. Curiously, even single amino acid substitutions within TM3 and TM4 led to poorly expressed protein, precluding their analysis. Nonetheless all other TM helices and loops were amenable to substitutions, allowing a dissection of the remainder of the TM domain. Each expressing construct was tested in our knockdown replacement assay and the results are quantified in Fig. 3A and schematized in Fig. 3B. (A full table of mutations and results is included in Table S1). The analysis revealed substantial stretches where residue identity was seemingly not important for function, such as all of TM1 and Loop2; however for others, such as TM2 and Loop1, substitutions of 7?0 amino acid stretches disrupted function. To further refine the required residues, we constructed point mutations within the seemingly required regions. Surprisingly, when variants with individual amino acid substitutions were produced, it became clear that only two point mutations, K146E and V152L, resulted in any loss of function, though the loss was modest (Fig. 4A, D). Given their proximity to one another, we also tested the effect of combining the two substitutions. Notably, the double mutant variant K146E/V152L was completely nonfuncDiscussion The residue identity of surprisingly few amino acids tested are important for the mechanism of Yip1A functionAs shown previously for its yeast counterpart in a viability assay [19], the majority of the cytoplasmic domain of Yip1A was dispensable for its ER structural maintenance function. This dispensable portion has no predicted secondary structure; therefore it is difficult to speculate on its potential role. Yet its persistence through evolution suggests a role, perhaps regulatory, under conditions yet-to-be assessed. In contrast to the cytoplasmic domain, we found that the membrane-spanning domain of Yip1AMutational A.
