SPE-39 Family Proteins Interact with the HOPS Complex and Function in Lysosomal Delivery

Abstract

Yeast and animal homotypic fusion and vacuole protein sorting (HOPS) complexes contain conserved subunits, but HOPS-mediated traffic in animals might require additional proteins. Here, we demonstrate that SPE-39 homologues, which are found only in animals, are present in RAB5-, RAB7-, and RAB11-positive endosomes where they play a conserved role in lysosomal delivery and probably function via their interaction with the core HOPS complex. Although Caenorhabditis elegans spe-39 mutants were initially identified as having abnormal vesicular biogenesis during spermatogenesis, we show that these mutants also have disrupted processing of endocytosed proteins in oocytes and coelomocytes. C. elegans SPE-39 interacts in vitro with both VPS33A and VPS33B, whereas RNA interference of VPS33B causes spe-39-like spermatogenesis defects. The human SPE-39 orthologue C14orf133 also interacts with VPS33 homologues and both coimmunoprecipitates and cosediments with other HOPS subunits. SPE-39 knockdown in cultured human cells altered the morphology of syntaxin 7-, syntaxin 8-, and syntaxin 13-positive endosomes. These effects occurred concomitantly with delayed mannose 6-phosphate receptor-mediated cathepsin D delivery and degradation of internalized epidermal growth factor receptors. Our findings establish that SPE-39 proteins are a previously unrecognized regulator of lysosomal delivery and that C. elegans spermatogenesis is an experimental system useful for identifying conserved regulators of metazoan lysosomal biogenesis. INTRODUCTIONLysosome biogenesis is mediated by multiple vesicular budding and fusion events that deliver components between subcellular compartments (Luzio et al., 2003). In Saccharomyces cerevisiae, vesicular fusion at the vacuole (the yeast equivalent of lysosomes) requires the homotypic fusion and vacuole protein sorting (HOPS) complex, which regulates vesicle docking through its interaction with soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) and the Rab protein Ypt7p Sato et al., 2000;Wurmser et al., 2000). The yeast HOPS complex contains class C Vps proteins Vps11p, Vps16p, Vps18p, and Vps33p (Rieder and Emr, 1997) and class B Vps proteins Vps39p and Vps41p (Seals et al., 2000;Wurmser et al., 2000). In addition to vesicular fusion at the vacuole, the class C Vps proteins also function in Golgi-to-endosome transport and other steps in vacuolar delivery pathways (Srivastava et al., 2000;Peterson and Emr, 2001;Subramanian et al., 2004;Peplowska et al., 2007). Involvement of the class C Vps proteins at multiple stages has also been reported in mammalian cells (Kim et al., 2003;Richardson et al., 2004). Vesicular trafficking to the lysosome has been extensively described in yeast (Bowers and Stevens, 2005), but it is also known that this process is more complex in metazoans (Dell'Angelica, 2004). Yeast only has lysosomes of a single type, whereas animals have lysosomes and lysosome-related organelles that are functionally diverse. This diversity is evident when comparing th...

Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

“…Published data regarding the role of VPS33B and VIPAS39 as members of the HOPS complex are seemingly conflicting (12,16). Our endogenous IP data (Fig.…”

Atg7 is required for acrosome biogenesis during spermatogenesis in mice

“…According to these similarities, the acrosome has been postulated to be a modified lysosome [4]. Components of the endocytic machinery, such as Afaf [14], SH3P13 [23], SPE-39 [24], UBPy [25], RasGRF1 [26], and Vps54 [27,28], are involved in the biogenesis of the acrosome [2]. This evidence strongly supports the notion that the acrosome is a novel LRO.…”

Inhibitory effect of SPE‐39 due to tyrosine phosphorylation and ubiquitination on the function of Vps33B in the EGF‐stimulated cells

“…Depletion of SPE-39 induced a delay in the downregulation of the EGF receptor 30 min after the addition of EGF. However, after several hours of EGF treatment, the amount of the EGF receptor was almost identical in cells with or without depletion of SPE-39 [4].…”

“…The HOPS complex comprises with four subunits of class C vacuolar protein sorting (Vps) proteins (Vps11, Vps16, Vps18 and Vps33) and two class B Vps proteins (Vps39 and Vps41) [2][3][4].…”

“…The human ortholog of SPE-39 (hSPE-39) is also known to associate with both subtypes of Vps33A and Vps33B in mammalian cells [4,7]. Furthermore, other research groups found hSPE-39 mutations, but not Vps33B mutations in individuals with ARC.…”