NBD-labeled phosphatidylcholine and phosphatidylethanolamine are internalized by transbilayer transport across the yeast plasma membrane

Traffic. 2001 Jan;2(1):37-50. doi: 10.1034/j.1600-0854.2001.020106.x.

Abstract

The internalization and distribution of fluorescent analogs of phosphatidylcholine (M-C6-NBD-PC) and phosphatidylethanolamine (M-C6-NBD-PE) were studied in Saccharomyces cerevisiae. At normal growth temperatures, M-C6-NBD-PC was internalized predominantly to the vacuole and degraded. M-C6-NBD-PE was internalized to the nuclear envelope/ER and mitochondria, was not transported to the vacuole, and was not degraded. At 2 degrees C, both were internalized to the nuclear envelope/ER and mitochondria by an energy-dependent, N-ethylmaleimide-sensitive process, and transport of M-C6-NBD-PC to and degradation in the vacuole was blocked. Internalization of neither phospholipid was reduced in the endocytosis-defective mutant, end4-1. However, following pre-incubation at 37 degrees C, internalization of both phospholipids was inhibited at 2 degrees C and 37 degrees C in sec mutants defective in vesicular traffic. The sec18/NSF mutation was unique among the sec mutations in further blocking M-C6-NBD-PC translocation to the vacuole suggesting a dependence on membrane fusion. Based on these and previous observations, we propose that M-C6-NBD-PC and M-C6-NBD-PE are transported across the plasma membrane to the cytosolic leaflet by a protein-mediated, energy-dependent mechanism. From the cytosolic leaflet, both phospholipids are spontaneously distributed to the nuclear envelope/ER and mitochondria. Subsequently, M-C6-NBD-PC, but not M-C6-NBD-PE, is sorted by vesicular transport to the vacuole where it is degraded by lumenal hydrolases.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenosine Triphosphatases*
  • Biological Transport
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism*
  • Endocytosis / drug effects
  • Endocytosis / physiology*
  • Enzyme Inhibitors / pharmacology
  • Ethylmaleimide / pharmacology
  • Flow Cytometry
  • Fluorescent Dyes / metabolism
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Intracellular Membranes / metabolism
  • Lipid Bilayers
  • Phosphatidylcholines / chemistry
  • Phosphatidylcholines / metabolism*
  • Phosphatidylethanolamines / chemistry
  • Phosphatidylethanolamines / metabolism*
  • Pyridinium Compounds / metabolism
  • Quaternary Ammonium Compounds / metabolism
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Sodium Azide / pharmacology
  • Sodium Fluoride / pharmacology
  • Temperature
  • Vacuoles / metabolism
  • Vesicular Transport Proteins*

Substances

  • Enzyme Inhibitors
  • FM 4-64
  • Fluorescent Dyes
  • Fungal Proteins
  • Lipid Bilayers
  • Phosphatidylcholines
  • Phosphatidylethanolamines
  • Pyridinium Compounds
  • Quaternary Ammonium Compounds
  • Saccharomyces cerevisiae Proteins
  • Vesicular Transport Proteins
  • Sodium Fluoride
  • Sodium Azide
  • Adenosine Triphosphatases
  • SEC18 protein, S cerevisiae
  • Ethylmaleimide