Abstract
We have investigated the effects of ligand concentration on the intracellular behavior of the yolk protein precursor vitellogenin in Xenopus oocytes. After internalization by receptor-mediated endocytosis, vitellogenin is delivered to a final storage compartment in mature, high density (1.23 g/ml sucrose) yolk platelets by a biphasic transport pathway. The first phase consists of a relatively rapid increase in the density of ligand-containing organelles to 1.21 g/ml, a region of the sucrose gradient containing a subpopulation of light yolk platelets and lysosomal enzyme activity. The second and rate-limiting phase in vitellogenin transport is the appearance of ligand in mature yolk platelets. The first phase of transport is accompanied by cleavage of vitellogenin into the mature yolk proteins. The majority of the light yolk platelets are not converted into heavy platelets when vitellogenin is absent from the medium for 24 h, indicating that they are a relatively stable organelle which does not mature into heavy platelets during this time period. The progress of vitellogenin through both phases of transport exhibits a concentration dependence, with much slower rates of density increase and lack of appearance in heavy yolk platelets at subsaturating ligand levels. The majority of vitellogenin entering oocytes at low ligand concentrations remains for at least 60 min in a low density endocytic compartment which is accessible to a second pulse of ligand. Vitellogenin-colloidal gold tracer studies indicate that this low density compartment is composed primarily of multivesicular bodies. Low concentrations of vitellogenin residing in this compartment for 4 h are no longer accessible to newly internalized ligand, suggesting that movement into an organelle incapable of fusing with endosomes has occurred.
Highlights
We have investigated theeffects of ligand concentra- lipid, approximately 1%carbohydrate, and approximately tion on the intracellular behavior of the yolk protein 1.4% phosphate by weight (Wallace, 1984)
The majority of the light yolk platelets are not con- tion and platelets arecompletely resorbed in a process which verted intoheavy platelets when vitellogenin is absent appears to involve lysosomal hydrolysis (Williams, 1967)
Examination of the specificity of this antiserum by immunoblotting (Hubbard et al.,1985; Roman and Hubbard, 1984)revealedthat theantibodies react strongly with SDSdenatured lipovitellin 2 (LV2) and relatively weakly with VG itself. These two proteins are readily distinguishable by SDS gel electrophoresis because of their size differences (31kDa for LV2 and 200 kDa for VG)
Summary
Protein determinations were performed according tothe Bio-Rad procedure supplied with the dye reagent concentrate, using bovine y-globulin as protein standard. Examination of the specificity of this antiserum by immunoblotting (Hubbard et al.,1985; Roman and Hubbard, 1984)revealedthat theantibodies react strongly with SDSdenatured LV2 and relatively weakly with VG itself These two proteins are readily distinguishable by SDS gel electrophoresis because of their size differences (31kDa for LV2 and 200 kDa for VG). 100pg of protein from each sucrose gradient fraction was run on 8-15% SDS-polyacrylamide gels, transferred to nitrocellulose (Towbin et al, 1979), and incubated with anti-LV2antiserum a t 1:3000 dilution, followed by '251-proteinA and autoradiography as described (Hubbard et al, 1985; Roman and Hubbard, 1984). Trace VG pulse experiments required that any exogenous proteins be omitted
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