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SNX12 role in endosome membrane transport.

Pons V, Ustunel C, Rolland C, Torti E, Parton RG, Gruenberg J - PLoS ONE (2012)

Bottom Line: We found that SNX12 is expressed at very low levels compared to SNX3.SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides.Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva, Switzerland.

ABSTRACT
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

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SNX12 is less expressed than SNX3 but is also localized on early endosomes.(A) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (B) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (C) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.
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pone-0038949-g001: SNX12 is less expressed than SNX3 but is also localized on early endosomes.(A) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (B) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (C) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.

Mentions: SNX12 (NCBI Reference Sequence: NM_013346.2) is a member of the sorting nexin protein family, which apparently contains a PX domain as sole structural domain. The protein consists of 162 amino acids with a predicted molecular weight of ∼20 kDa. Alignment reveals that SNX12 and SNX3, which is also a PX-only protein, share the highest identity amongst SNX family members, with 70.8% DNA identity and 79.5% protein identity (Figure 1A). Since we previously studied the role of SNX3 in protein sorting and membrane transport, we investigated whether SNX12 could also share the same functions as SNX3. To this end, we first quantified by quantitative PCR the relative amount of SNX12 and SNX3 transcripts in different cell lines such as HeLa, HepG2, Caco-2 and PC-3 cells. We found that in these cell lines, SNX12 is expressed at very low levels when compared to SNX3 (≈100–200 fold less) (Figure 1B). This is in good agreement with an exhaustive analysis of the transcription profiles of SNX3 and SNX12 in tissues and cell lines carried out with Genevestigator (https://www.genevestigator.com/gv/index.jsp), which combines thousands of microarray experiments. Interestingly, SNX3 and SNX12 genes are highly conserved in vertebrates. However, only one common ancestor gene is found in insects (D. melanogaster) and yeast (S. cerevisiae), suggesting a gene duplication during evolution. In human, it is important to note that SNX3 gene is located on chromosome 6 (position 6q21) whereas SNX12 gene is located on chromosome X (location Xq13.1). It is thus possible that the expression of the SNX12 gene on sex chromosome is more tightly regulated than that of SNX3 on an autosomal chromosome.


SNX12 role in endosome membrane transport.

Pons V, Ustunel C, Rolland C, Torti E, Parton RG, Gruenberg J - PLoS ONE (2012)

SNX12 is less expressed than SNX3 but is also localized on early endosomes.(A) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (B) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (C) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376135&req=5

pone-0038949-g001: SNX12 is less expressed than SNX3 but is also localized on early endosomes.(A) Alignment of amino acid sequences of Homo sapiens SNX3 and SNX12. (B) RNA was extracted from different cell lines as indicated and relative amounts of endogenous SNX3 and SNX12 mRNA were quantified by RT-PCR. Values are indicated in the table below the graph since they are very low for SNX12 mRNA. (C) HeLa cells expressing GFP-SNX12 or co-expressing GFP-SNX12 and mRFP1-SNX3 were processed for immunofluorescence using the indicated antibodies. Scale bar indicates 10 µm.
Mentions: SNX12 (NCBI Reference Sequence: NM_013346.2) is a member of the sorting nexin protein family, which apparently contains a PX domain as sole structural domain. The protein consists of 162 amino acids with a predicted molecular weight of ∼20 kDa. Alignment reveals that SNX12 and SNX3, which is also a PX-only protein, share the highest identity amongst SNX family members, with 70.8% DNA identity and 79.5% protein identity (Figure 1A). Since we previously studied the role of SNX3 in protein sorting and membrane transport, we investigated whether SNX12 could also share the same functions as SNX3. To this end, we first quantified by quantitative PCR the relative amount of SNX12 and SNX3 transcripts in different cell lines such as HeLa, HepG2, Caco-2 and PC-3 cells. We found that in these cell lines, SNX12 is expressed at very low levels when compared to SNX3 (≈100–200 fold less) (Figure 1B). This is in good agreement with an exhaustive analysis of the transcription profiles of SNX3 and SNX12 in tissues and cell lines carried out with Genevestigator (https://www.genevestigator.com/gv/index.jsp), which combines thousands of microarray experiments. Interestingly, SNX3 and SNX12 genes are highly conserved in vertebrates. However, only one common ancestor gene is found in insects (D. melanogaster) and yeast (S. cerevisiae), suggesting a gene duplication during evolution. In human, it is important to note that SNX3 gene is located on chromosome 6 (position 6q21) whereas SNX12 gene is located on chromosome X (location Xq13.1). It is thus possible that the expression of the SNX12 gene on sex chromosome is more tightly regulated than that of SNX3 on an autosomal chromosome.

Bottom Line: We found that SNX12 is expressed at very low levels compared to SNX3.SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides.Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Geneva, Geneva, Switzerland.

ABSTRACT
In this paper, we investigated the role of sorting nexin 12 (SNX12) in the endocytic pathway. SNX12 is a member of the PX domain-containing sorting nexin family and shares high homology with SNX3, which plays a central role in the formation of intralumenal vesicles within multivesicular endosomes. We found that SNX12 is expressed at very low levels compared to SNX3. SNX12 is primarily associated with early endosomes and this endosomal localization depends on the binding to 3-phosphoinositides. We find that overexpression of SNX12 prevents the detachment (or maturation) of multivesicular endosomes from early endosomes. This in turn inhibits the degradative pathway from early to late endosomes/lysosomes, much like SNX3 overexpression, without affecting endocytosis, recycling and retrograde transport. In addition, while previous studies showed that Hrs knockdown prevents EGF receptor sorting into multivesicular endosomes, we find that overexpression of SNX12 restores the sorting process in an Hrs knockdown background. Altogether, our data show that despite lower expression level, SNX12 shares redundant functions with SNX3 in the biogenesis of multivesicular endosomes.

Show MeSH
Related in: MedlinePlus