Inverting the ALPS motif sequence does not affect the capacity of GMAP_N to accumulate early secretory pathway vesicles. (A) The GMAP-210 ALPS motif sequence and its inverted sequence (top) and their helical wheel representations (bottom). Yellow: Val, Leu, Ile, Met, Phe, and Trp. Pink: Ser and Thr. Red: Asp and Glu. Blue: Lys and Arg. Grey: other residues. (B) Representative EM image of cells expressing invALPS-GMAP. Inset shows boxed region at a higher magnification. N, nucleus; V, vacuole; CW, cell wall. (C) Localization of invALPS-GMAP–mCherry with GFP-Bet1p after 4 h of induction (top) and of invALPS-GMAP–GFP with FM4-64 after 2.5 h of induction (bottom). Bar, 2 µm. (D) Quantification of localization of invALPS-GMAP–mCherry with GFP-Bet1p, GFP-Sec22p, or GFP-Emp47p or of invALPS-GMAP–GFP with FM4-64 at the indicated times after induction. SEY6210 (or BY4742 for FM4-64 imaging) cells carrying the indicated proteins on plasmids were grown overnight under inducing conditions (B) or overnight under repressing conditions and shifted to induction medium for 2.5 or 4 h (C and D). Means and SDs of at least three independent experiments are shown. Outlines of cells are indicated by dashed lines. N, N terminus; C, C terminus.