Abstract
The cellular endomembrane system requires the proper kinetic balance of synthesis and degradation of its individual components, which is maintained in part by a specific membrane fusion apparatus. In this study, we describe the molecular properties of D12, which was identified from a mouse expression library. This C-terminal anchored membrane protein has sequence similarity to both a yeast soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE), Use1p/Slt1p, and a recently identified human syntaxin 18-binding protein, p31. D12 formed a tight complex with syntaxin 18 as well as Sec22b and bound to alpha-SNAP, indicating that D12 is a SNARE protein. Although the majority of D12 is located in the endoplasmic reticulum and endoplasmic reticulum-Golgi intermediate compartments at steady state, overexpression or knockdown of D12 had no obvious effects on membrane trafficking in the early secretory pathway. However, suppression of D12 expression caused rapid appearance of lipofuscin granules, accompanied by apoptotic cell death without the apparent activation of the unfolded protein response. The typical cause of lipofuscin formation is the impaired degradation of mitochondria by lysosomal degradative enzymes, and, consistent with this, we found that proper post-Golgi maturation of cathepsin D was impaired in D12-deficient cells. This unexpected observation was supported by evidence that D12 associates with VAMP7, a SNARE in the endosomal-lysosomal pathway. Hence, we suggest that D12 participates in the degradative function of lysosomes.
Highlights
Specific fusion of biological membranes is required for many cellular processes, including membrane trafficking between different organelles and within individual organelles, and is executed in eukaryotic cells by fusogenic soluble N-ethylmaleimide-sensitive factor (NSF)3 attachment protein (SNAP) receptors (SNAREs) [1]
All SNAREs are characterized by homologous stretches of 60 –70 amino acids referred to as SNARE motifs, which are adjacent to the membrane anchor domains
We show here that D12 is a Q-SNARE and, whereas it is mostly localized in the ER and ERGIC, it binds to VAMP7, a SNARE involved in endosome-lysosome transport
Summary
Specific fusion of biological membranes is required for many cellular processes, including membrane trafficking between different organelles and within individual organelles, and is executed in eukaryotic cells by fusogenic soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptors (SNAREs) [1]. Use1p/Slt1p was identified as a novel SNARE located in the ER. This is an essential protein and is thought to function in retrograde transport from the Golgi to the ER, because Use1p/Slt1p is associated with the retrograde SNAREs Ufe1p, Sec22p, and Sec20p but not with the anterograde SNAREs Bos1p and Bet1p [7, 8]. BNIP1 and p31, which have similarity to Sec20p and Use1p/Slt1p in yeast, were identified as syntaxin 18-associated proteins. Our analyses indicate that D12 plays no role in membrane trafficking in the early secretory pathway but rather is involved in the maintenance of proper lysosomal function
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