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. 2005 Oct 18;102(42):15173-7.
doi: 10.1073/pnas.0409558102. Epub 2005 Oct 7.

Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2

Courtney T Griffin  1 , JoAnn Trejo, Terry Magnuson
Affiliations

Genetic evidence for a mammalian retromer complex containing sorting nexins 1 and 2

Courtney T Griffin et al. Proc Natl Acad Sci U S A. .

Abstract

We have previously shown that the putative mammalian retromer components sorting nexins 1 and 2 (Snx1 and Snx2) result in embryonic lethality when simultaneously targeted for deletion in mice, whereas others have shown that Hbeta58 (also known as mVps26), another retromer component, results in similar lethality when targeted for deletion. In the current study, we address the genetic interaction of these mammalian retromer components in mice. Our findings reveal a functional interaction between Hbeta58, SNX1, and SNX2 and strongly suggest that SNX2 plays a more critical role than SNX1 in retromer activity during embryonic development. This genetic evidence supports the existence of mammalian retromer complexes containing SNX1 and SNX2 and identifies SNX2 as an important mediator of retromer biology. Moreover, we find that mammalian retromer complexes containing SNX1 and SNX2 have an essential role in embryonic development that is independent of cation-independent mannose 6-phosphate receptor trafficking.

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Figures

Fig. 1.
Fig. 1.
Snx2 mRNA is more abundant than Snx1 mRNA in extraembryonic yolk sacs at midgestation. (A) RT-PCR. Snx1, Snx2, and Hβ58 were amplified by RT-PCR from a litter of E8.5 wild-type embryos or their yolk sacs. (-RT) indicates mock RT reactions in which no reverse transcriptase was used. Equal amounts of +/-RT templates were used in each PCR, and identical amplification conditions were used for all samples. This experiment was repeated three times, and the quantitative averages of the data reveal that Snx2 mRNA is expressed at ≈72% of Snx1 mRNA levels in the embryo, but Snx2 mRNA levels are ≈225% of Snx1 mRNA levels in the yolk sac. (B) PCR amplification of Snx1 and Snx2 cDNA for assessing primer pair amplification ability. Snx1 or Snx2 cDNAs (0.15 ng) were PCR amplified with their gene-specific primers for varying numbers of cycles to demonstrate the comparable ability of the primers to amplify equal amounts of template.
Fig. 2.
Fig. 2.
CI-MPR sublocalization and stability is unaltered in wild-type versus retromer-deficient MEF cell lines. (A) CI-MPR/EEA1 colocalization. Low passage, primary wild-type and Snx1-/-;Snx2-/- MEF lines were fixed and stained for endogenous CI-MPR (green) and EEA1 (red) and imaged by confocal microscopy. Insets are magnifications of boxed areas, and arrowheads indicate coincident labeling. (B) CI-MPR protein levels in Snx1-/-, Snx2-/-, and Snx1-/-;Snx2-/- MEFs. High passage, immortalized MEF lines were either left untreated or were treated with 40 μg/ml cycloheximide diluted in αMEM for 17 h. Cell lysates were then immunoblotted with an anti-CI-MPR antibody, followed by an anti-actin antibody to control for equal loading. (C) Quantification of CI-MPR stability in Snx1-/-, Snx2-/-, and Snx1-/-;Snx2-/- MEFs. Three separate experiments, such as those shown in B, were quantified, and the data (mean ± SEM) are expressed as a percentage of CI-MPR remaining (dark gray bars) compared with untreated controls (light gray bars).
Fig. 3.
Fig. 3.
CI-MPR localization is unaltered in control versus mutant extraembryonic tissues. (A–D) CI-MPR localization in E8.5 extraembryonic yolk sacs. Hβ58+/- versus Hβ58-/- and Snx1-/- versus Snx1-/-;Snx2-/- littermate embryos were dissected and genotyped, whereas their yolk sacs were subjected to whole mount immunofluorescence analysis with an anti-CI-MPR antibody (green). Yolk sacs were mounted on glass slides with mounting media containing DAPI (blue) before imaging. (Magnification: ≈×40.) (E–H) CI-MPR localization in E6.5 visceral endoderm cells. Visceral endoderm was separated from the epiblasts of Snx1-/- versus Snx1-/-;Snx2-/- and Hβ58+/+ versus Hβ58-/- littermate embryos at E6.5. The embryos were genotyped, whereas clumps of visceral endoderm cells were cytospun onto glass slides and subjected to immunofluorescence analysis with an anti-CI-MPR antibody (green) and mounting media containing DAPI (blue) before imaging. (Magnification: ≈×100.)
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