The pathway and targeting signal for delivery of the integral membrane glycoprotein LEP100 to lysosomes.
A complete set of chimeras was made between the lysosomal membrane glycoprotein LEP100 and the plasma membrane-directed vesicular stomatitis virus G protein, combining a glycosylated lumenal or ectodomain, a single transmembrane domain, and a cytosolic carboxyl-terminal domain. These chimeras, the parent molecules, and a truncated form of LEP100 lacking the transmembrane and cytosolic domains were expressed in mouse L cells. Only LEP100 and chimeras that included the cytosolic 11 amino acid carboxyl terminus of LEP100 were targeted to lysosomes. The other chimeras accumulated in the plasma membrane, and truncated LEP100 was secreted. Chimeras that included the extracellular domain of vesicular stomatitis G protein and the carboxyl terminus of LEP100 were targeted to lysosomes and very rapidly degraded. Therefore, in chimera-expressing cells, virtually all the chimeric molecules were newly synthesized and still in the biosynthesis and lysosomal targeting pathways. The behavior of one of these chimeras was studied in detail. After its processing in the Golgi apparatus, the chimera entered the plasma membrane/endosome compartment and rapidly cycled between the plasma membrane and endosomes before going to lysosomes. In pulse-expression experiments, a large population of chimeric molecules was observed to appear transiently in the plasma membrane by immunofluorescence microscopy. Soon after protein synthesis was inhibited, this surface population disappeared. When lysosomal proteolysis was inhibited, chimeric molecules accumulated in lysosomes. These data suggest that the plasma membrane/early endosome compartment is on the pathway to the lysosomal membrane. This explains why mutations that block endocytosis result in the accumulation of lysosomal membrane proteins in the plasma membrane.
Mathews
P M
PM
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218.
Martinie
J B
JB
Fambrough
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D030161:Avian Proteins; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D007739:L Cells; D008247:Lysosomes / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014759:Viral Envelope Proteins / Q000378:metabolism
D030161:Avian Proteins; C028391:G protein, vesicular stomatitis virus; D008562:Membrane Glycoproteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C066541:LEP100 protein, Gallus gallus; D003513:Cycloheximide