Developmentally regulated trafficking of the lysosomal membrane protein p67 in Trypanosoma brucei.
p67 is a lysosomal type I membrane glycoprotein of Trypanosoma brucei. In procyclic stage cells p67 trafficks to the lysosome without modification, but in the bloodstream stage Golgi processing adds poly-N-acetyllactosamine to N-glycans. In both stages proteolytic fragmentation occurs in the lysosome, but turnover is approximately nine times faster in bloodstream cells. Trafficking of wildtype p67 and mutants missing the cytoplasmic (p67DeltaCD) or cytoplasmic/transmembrane domains (p67DeltaTM) was monitored by pulse-chase, surface biotinylation and immunofluorescence. Overexpressed wildtype p67 trafficks normally in procyclics, but some leaks to the cell surface suggesting that the targeting machinery is saturable. p67DeltaCD and p67DeltaTM are delivered to the cell surface and secreted, respectively. The membrane/cytoplasmic domains function correctly in procyclic cells when fused to GFP indicating that these domains are sufficient for stage-specific lysosomal targeting. In contrast, p67 wildtype and deletion reporters are overwhelmingly targeted to the lysosome and degraded in bloodstream cells. These findings suggest that either redundant developmentally regulated targeting signals/machinery are operative in this stage or that the increased endocytic activity of bloodstream cells prevents export of the deletion reporters.
Alexander
David L
DL
The Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, WI 53706, USA.
Schwartz
Kevin J
KJ
Balber
Andrew E
AE
Bangs
James D
JD
D000595:Amino Acid Sequence; D000818:Animals; D004705:Endocytosis / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D030781:Organisms, Genetically Modified; D021381:Protein Transport / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism
D008565:Membrane Proteins; D011506:Proteins; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; C018589:lysosomal proteins