A tubular endosomal fraction from rat liver: biochemical evidence of receptor sorting by default - PubMed (original) (raw)

A tubular endosomal fraction from rat liver: biochemical evidence of receptor sorting by default

M Vergés et al. Proc Natl Acad Sci U S A. 1999.

Abstract

We previously have isolated an endosomal fraction from rat liver, termed receptor-recycling compartment (RRC), which is highly enriched in recycling receptors and in the transcytotic polymeric Ig receptor (pIgR). We now have analyzed the RRC fraction by immunoisolation and found that no uniquely transcytotic elements were present, because recycling receptors and the pIgR were coisolated on the same elements. In addition, RRC was very rich in proteins previously shown to be associated with recycling endosomes, such as rab 11, cellubrevin, and endobrevin, but relatively poor in early endosome antigen 1. As RRC contains mainly tubules and small vesicles, our results indicate that it is enriched in elements of a tubular endosomal compartment involved in receptor sorting. Biochemical analysis showed that the density of recycling receptors and transcytotic pIgR in RRC membranes was similar to that in early endosome membranes. This observation supports the idea that increasing membrane surface area by endosome tubulation is the main mechanism to ensure efficient receptor sorting and, at the same time, locates RRC in a common step of the endocytotic system before final receptor segregation into distinct recycling and transcytotic pathways.

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Figures

Figure 1

Figure 1

Representative Western blots showing distribution of proteins in the endosome fractions and enrichment over the liver homogenate. The same amount of protein (3–10 μg, depending on the antigen) was loaded on each of the compared lanes of endosome fractions. The values are from quantifications by densitometric scanning of Western blots and indicate the fold of protein enrichment, in each fraction, over the liver homogenate. The reported value corresponds to the mean ± SEM of ≥ 3 different experiments, except for Tf (two).

Figure 2

Figure 2

Immunoisolations of RRC show that distinct transcytotic elements were not detected; rather, recycling receptors and the pIgR were coisolated on the same elements. All immunoisolations were carried out by using 5 μg of RRC protein per 1 mg of antibody-coated magnetic beads. The entire sample from each population of elements was loaded onto the gels. (A) Immunoisolation (ImIs) was carried out by using magnetic beads coated with the anti-pIgR SC166 antibody. (B) The first immunoisolation (ImIs.1) was done by using magnetic beads coated with the anti-LDLR 4A4 antibody. The second immunoisolation (ImIs.2) was done by using magnetic beads coated with the anti-pIgR SC166 antibody. Starting material (SM), bound (B), and unbound (UB) populations of elements were analyzed by Western blotting using either the SC166 antibody or the antibodies against the recycling receptors, LDLR and TfR.

Figure 3

Figure 3

Representative Western blots showing distribution of proteins involved in membrane traffic in the endosome fractions and enrichment over the liver homogenate. The same amount of protein (3–10 μg, depending on the antigen) was loaded on each of the compared lanes of endosome fractions. The values are from quantifications by densitometric scanning of Western blots and indicate the fold of protein enrichment, in each fraction, over the liver homogenate. The reported value corresponds to the mean ± SEM of ≥ 3 different experiments. EEA1, early endosome antigen 1.

Figure 4

Figure 4

Representative Western blots showing distribution of the transcytotic pIgR, the recycling receptors, LDLR and TfR, and apo B-100 (LDL) in intact endosome fractions and in endosome membranes. The same amount of protein (1 μg) was loaded on each lane. Endosome membranes were isolated by means of high-pH sodium carbonate treatment, by which 60–70% of the contents was removed.

Figure 5

Figure 5

Graphic representations showing the density of receptors versus the percentage of LDL remaining in the membranes. The x axis is the % apo B-100 (LDL) remaining in the membranes (m), taking the intact (noncarbonate stripped) fraction (i) as 100% for each endosome fraction. The y axis is the relative density of each receptor, expressed as fold enrichment over the total homogenate.

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