Phosphatidylserine is a component of the gradient-tracking machine in mating yeast.

Chemotropism, the ability to orient growth toward external chemical cues, is a fundamental process in diverse eukaryotic systems. During mating, budding yeast cells detect pheromone gradients from potential partners, locating them by assembling a gradient-tracking machine (GTM) at the plasma membrane that redistributes upgradient prior to polarized growth. Although membrane lipids are known to influence pheromone signaling and morphogenesis, their roles in pre-morphogenic gradient tracking have remained unclear. Here, we show that phosphatidylserine (PS), phosphatidylinositol-4,5-bisphosphate, and ergosterol exhibit GTM-like dynamics, polarizing to the default polarity site, redistributing upgradient, and stabilizing at the chemotropic site. Blocking PS synthesis causes a severe and specific gradient-tracking defect, whereas disruption of Bem1 binding to anionic lipids slows but does not abolish tracking. Analysis of polarity, Cdc42 activity, and exocyst dynamics indicates that Bem1 membrane binding contributes to spatial focusing of Cdc42 activation during tracking but cannot account for the pronounced defects caused by PS loss, indicating that PS influences gradient tracking through multiple GTM components. In contrast, ergosterol is dispensable for tracking but required for proper receptor organization and partner alignment after GTM stabilization. Together, these findings establish membrane lipids as integral GTM components and highlight PS as a key regulator of chemotropic gradient sensing through multivalent protein-lipid interactions.