LDL transcytosis passes through the trans-Golgi network and requires Rab10

Atherosclerosis begins with the subendothelial retention of low-density lipoproteins (LDL) from the circulation. While LDL transcytosis across the endothelium is mediated by SR-BI and ALK1 and is usually independent of LDLR, the intracellular mechanisms and route of LDL transcytosis remain unclear. Using total internal reflection fluorescence microscopy in LDLR-depleted human coronary artery endothelial cells (HCAECs), we found that LDL transcytosis can proceed both directly as well as indirectly from an intracellular compartment. During LDL transcytosis, LDL was observed to colocalize with the Golgi apparatus over time, specifically with the trans-Golgi network marker TGN46. Systematic examination of endothelial Rab proteins known to regulate Golgi traffic identified Rabs6a and 10 to be required for LDL transcytosis. Depletion of Rab10 or Rab6a significantly inhibited LDL transcytosis but had no effect on albumin transcytosis. Expression and localization of SR-BI and ALK1 were also unimpaired. Conversely, over-expression of Rab10 increased LDL transcytosis. Finally, depletion of Rab10 increased colocalization of LDL with the trans-Golgi network and led to expansion of the Golgi, indicative of impaired exocytosis from the Golgi. However, colocalization of Rab10 with LDL did not increase over time and Rab10 did not accumulate at the base of the cell, suggesting its role is specifically related to LDL exit from the Golgi rather than direct transport. In summary, during LDL transcytosis, internalized LDL is transported to the Golgi where it serves as a reservoir of LDL that can undergo exocytosis. Our results identify specific Rab proteins as critical regulators of this process.