O support Yip1A function. This was intriguing given that the yeast mutant counterpart of K146E (K130E) was similarly disruptive to Yip1p function when combined with another mutation [19]. Importantly, we confirmed that the loss of function of these and other key nonrescuing variants described above was not due to low protein expression levels (Fig. S1). In sum, though multiple large-scale substitutions within the TM domain of Yip1A were disruptive, the identity of only a few individual residues, namely K146 in the first luminal loop, and V152 ?a nearby residue at the start of the second TM helix ?were clearly necessary for 
regulation of ER whorl formation.Required residues in Yip1A may control ER whorl formation independently of its established binding partners Yif1A/Yif1p and the Ypt1p/Ypt31p subclass of GTPasesIt was initially surprising that our unbiased analysis revealed in essence only two discrete sites crucial for the ER structural maintenance function of Yip1A (residues centered around E95 and K146). However it was also satisfying that the identified sites corresponded precisely to two sites previously shown to be essential for Yip1p-dependent viability in yeast (E76 and K130). More surprising was our finding that a third site previously shown to be essential for Yip1p function in yeast (E70) was completely dispensable for the control of ER whorl formation by Yip1A. This suggested that Yip1A/Yip1p might possess two separate functions. One function ?supported by E95 (E76 in yeast) and its flanking residues L92 and L96, as well as K146 (K130 in yeast) and nearby residue V152 ?that is required for ER structural maintenance; and another, supported by E89 (E70), that is dispensable for ER structural maintenance. Furthermore, the ability of Yip1p to bind its established binding partners Yif1p and Ypt1p/Ypt31p, mapped to E70 [19], the residue dispensable for control for ER whorl formation by Yip1A. Thus it seemed that the ER structuring function of Yip1A/Yip1p might operate independently 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 JSI-124 supplier depend on Yif1A, we reproduced the previously published Yif1A knockdown but with 1662274 an eye towards 15755315 revealing any potential ER phenotypes. Consistent with a lack of requirement for Yif1A in ER structural maintenance, we Terlipressin 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 revers.O support Yip1A function. This was intriguing given that the yeast mutant counterpart of K146E (K130E) was similarly disruptive to Yip1p function when combined with another mutation [19]. Importantly, we confirmed that the loss of function of these and other key nonrescuing variants described above was not due to low protein expression levels (Fig. S1). In sum, though multiple large-scale substitutions within the TM domain of Yip1A were disruptive, the identity of only a few individual residues, namely K146 in the first luminal loop, and V152 ?a nearby residue at the start of the second TM helix ?were clearly necessary for regulation of ER whorl formation.Required residues in Yip1A may control ER whorl formation independently of its established binding partners Yif1A/Yif1p and the Ypt1p/Ypt31p subclass of GTPasesIt was initially surprising that our unbiased analysis revealed in essence only two discrete sites crucial for the ER structural maintenance function of Yip1A (residues centered around E95 and K146). However it was also satisfying that the identified sites corresponded precisely to two sites previously shown to be essential for Yip1p-dependent viability in yeast (E76 and K130). More surprising was our finding that a third site previously shown to be essential for Yip1p function in yeast (E70) was completely dispensable for the control of ER whorl formation by Yip1A. This suggested that Yip1A/Yip1p might possess two separate functions. One function ?supported by E95 (E76 in yeast) and its flanking residues L92 and L96, as well as K146 (K130 in yeast) and nearby residue V152 ?that is required for ER structural maintenance; and another, supported by E89 (E70), that is dispensable for ER structural maintenance. Furthermore, the ability of Yip1p to bind its established binding partners Yif1p and Ypt1p/Ypt31p, mapped to E70 [19], the residue dispensable for control for ER whorl formation by Yip1A. Thus it seemed that the ER structuring function of Yip1A/Yip1p might operate independently 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 1662274 an eye towards 15755315 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 revers.
