TMEM74, a lysosome and autophagosome protein, regulates autophagy

https://doi.org/10.1016/j.bbrc.2008.02.055Get rights and content

Abstract

Autophagy is an intracellular degradation/recycling process in eukaryotic cells. It contributes to the turnover of cellular components by delivering portions of the cytoplasm and organelles to lysosomes for digestion. The molecular mechanisms of autophagy and vesicle trafficking, especially the biogenesis and turnover of autophagosomes, are poorly understood. In this report, we describe the biological activity of a novel autophagy-related molecule, FLJ30668, or Transmembrane protein 74 (TMEM74). Its transcript was identified by Northern blot and the open reading frame was found to encode 393 amino acids, which shared very little identity with other genetic products. Subcellular localization analysis showed TMEM74 localized to the lysosome and autophagosome. Overexpression of TMEM74 in HeLa cells resulted in autophagic vacuolization, increased the dotted distribution of MDC and GFP-LC3, and endogenous LC3-II levels. Wortmannin, an autophagy inhibitor, partially attenuated these effects. Moreover, knockdown of TMEM74 by small interference RNA abolished the autophagic characteristics induced by starvation. These findings demonstrate that TMEM74 may be involved in promoting functional autophagy during cell starvation and other stress conditions.

Section snippets

Materials and methods

Materials. Earle’s balanced salt solution (EBSS), bafilomycin A1, monodansylcadaverine (MDC), and a monoclonal antibody against β-actin were obtained from Sigma (USA). cDNA libraries of cancer tissues were obtained from Shanghai Genomics, Inc. Antibodies against cathepsinD and MAP-LC3 were purchased from Santa Cruz (Santa Cruz Biotechnology, Inc.). LysoTracker Red was obtained from Molecular Probes (USA). ER-DsRed plasmid was obtained from Clontech (USA). Alexa Fluor 780-labeled IgG secondary

Cloning, bioinformatics analysis and expression profile of human TMEM74

The human TMEM74 cDNA clone [15] was directly isolated from a human esophagus cDNA library and found to be 2087 base pairs long with an in-frame stop codon upstream of the putative ATG start codon. The sequence surrounding the ATG start codon largely followed the Kozak consensus rule. The open reading frame encoded 305 amino acids with a predicted molecular mass of 33.33 kDa and an isoelectric point of 4.87. The full-length cDNA and predicted amino acid sequence of TMEM74 are shown in Fig. 1A.

Discussion

Our data suggest that TMEM74 induces autophagic characters of cells, including cytoplasmic vacuolization under TEM (Fig. 2A), punctate distribution of GFP-LC3 (Fig. 2B) and MDC staining (Fig. 2D), and conversion of LC3-I to LC3-II (Fig. 2E). The autophagic response requires delivery of the contents sequestered by the autophagosome to the lysosome, whereupon degradation occurs [18]. Cytoplasmic vacuolization might reflect an increase in autophagic activity or a build-up of nonfused

Acknowledgments

Contract grant sponsor: China High Tech 863 Program; Contract Grant No. 2006AA02A305.

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