Effects of Sterol Structure and Sterol Ability to form Ordered Membrane Domains upon Cellular Endocytosis

Abstract

Ordered membrane domains (lipid rafts) have been hypothesized to participate in many cellular processes, including post-golgi trafficking, endocytosis, membrane protein sorting, cell viability and pathogen infection. In many cases, the evidence consists of an effect of the partial removal or sequestering of cholesterol from plasma membranes, based on the assumption that this disrupts formation of lipid domains. However, partial cholesterol removal does not establish that membrane domains were disrupted, and does not rule out a direct effect of cholesterol not related to domain formation. Thus, to investigate the role of the sterol structure and sterol raft-forming ability upon endocytosis, we used a sterol-substitution strategy. To do this, cyclodextrin-catalyzed lipid exchange was used to replace cholesterol with each of 16 sterols having various raft-forming abilities and variable structures. Endocytosis of two different proteins was studied - clustered placental alkaline phosphatase (PLAP)-endocytosis (clathrin-independent) and transferrin endocytosis (clathrin-mediated). Both types of endocytosis were affected similarly by sterol type. The ability to support lipid raft formation was found to be necessary for endocytosis. However, it was not sufficient for endocytosis, because sterols/steroids in which the 3β-OH group of cholesterol was modified did not support endocytosis. The loss of endocytosis could be reversed by a second substitution step that replenished cholesterol, and although some sterols that do not support ordered domain formation altered cell shape, cell shape changes were not found to be closely linked to the loss of endocytosis. This study shows the sterol-substitution method using diverse sterols is a useful approach to study the potential role of rafts and sterols in plasma membrane on cellular functions.