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Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference

Biological Procedures Online volume 7, pages 17–25 (2005)Cite this article

Abstract

To assess the role of sortilin in the sorting and trafficking of sphingolipid activator proteins (SAPs) the function of sortilin was abolished by a dominant-negative mutant and by the use of RNAi. Mutant sortilin lacking the carboxyl-terminal region that contains the sorting signal abolished the trafficking of SAPs to the lysosomes. Both sortilin and SAPs were retained in the Golgi apparatus. The use of chemically synthesized siRNA effectively blocked the trafficking of SAPs to the lysosomes as well. Additionally, we created a stable COS-7 cell line transfected with the pSilencer 3.1 H1 neo vector containing a selected siRNA template oligonucleotide (small hairpin interference RNA) where the levels of sortilin were greatly suppressed. The elimination of sortilin by this method will permit to determine whether or not sortilin is involved in a general mechanism of lysosomal sorting that involves the trafficking of various soluble lysosomal proteins other than SAPs.

Abbreviations

GM2AP:

GM2 activator protein

LAMP:

lysosomal associated protein

LIMP:

lysosomal integral membrane protein-1

M6P-R:

mannose 6-phosphate receptor

RNAi:

RNA interference

SAP:

sphingolipid activator protein

siRNA:

small interference RNA

shRNA:

small hairpin interference RNA

AP-1:

Adaptor Protein-1

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Authors and Affiliations

  1. Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada

    Stephane Lefrancois, Maryssa Canuel, Jibin Zeng & Carlos R. Morales

Authors
  1. Stephane Lefrancois
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  2. Maryssa Canuel
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  3. Jibin Zeng
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  4. Carlos R. Morales
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Correspondence to Stephane Lefrancois.

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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lefrancois, S., Canuel, M., Zeng, J. et al. Inactivation of sortilin (a novel lysosomal sorting receptor) by dominant negative competition and RNA interference. Biol. Proced. Online 7, 17–25 (2005). https://doi.org/10.1251/bpo101

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  • DOI: https://doi.org/10.1251/bpo101

Indexing terms

  • Sortilin
  • Sphingolipid Activator Proteins

Biological Procedures Online

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