Di-Leucine Signals Mediate Targeting of Tyrosinase and Synaptotagmin to Synaptic-like Microvesicles within PC12 Cells

Abstract

One pathway in forming synaptic-like microvesicles (SLMV) involves direct budding from the plasma membrane, requires adaptor protein 2 (AP2) and is brefeldin A (BFA) resistant. A second route leads from the plasma membrane to an endosomal intermediate from which SLMV bud in a BFA-sensitive, AP3-dependent manner. Because AP3 has been shown to bind to a di-leucine targeting signal in vitro, we have investigated whether this major class of targeting signals is capable of directing protein traffic to SLMV in vivo. We have found that a di-leucine signal within the cytoplasmic tail of human tyrosinase is responsible for the majority of the targeting of HRP-tyrosinase chimeras to SLMV in PC12 cells. Furthermore, we have discovered that a Met-Leu di-hydrophobic motif within the extreme C terminus of synaptotagmin I supports 20% of the SLMV targeting of a CD4-synaptotagmin chimera. All of the traffic to the SLMV mediated by either di-Leu or Met-Leu is BFA sensitive, strongly suggesting a role for AP3 and possibly for an endosomal intermediate in this process. The differential reduction in SLMV targeting for HRPtyrosinase and CD4-synaptotagmin chimeras by di-alanine substitutions or BFA treatment implies that different proteins use the two routes to the SLMV to differing extents. INTRODUCTIONThe efficient sorting of many transmembrane proteins to a variety of post-Golgi destinations is controlled by short specific sequences located within their cytoplasmic domains, sorting signals (for review, see Trowbridge et al., 1993;Sandoval and Bakke, 1994). At present, two major groups of sorting signals have been identified. The first group comprises tyrosine-based signals, which usually conform to the consensus YXXØ (where X is any amino acid, and Ø is a strong hydrophobic amino acid) or FXNPXY. The second group of sorting signals contains di-leucine/di-hydrophobic signals, in which one of the leucines can be substituted by isoleucine, methionine, or valine without loss of function (Letourner and Klausner, 1992;Bremnes et al., 1994;Sandoval and Bakke, 1994;Pond et al., 1995). Sorting signals falling outside these groups include the amphipathic ␣-helixes, which can adopt a supercoiled conformation and were found in the cytoplasmic domains of vesicle-associated membrane protein II (VAMPII) and the ␤-chain of the interleukin-1 receptor (Grote et al., 1995;Subtil et al., 1997). In addition, clusters of acidic residues in the context of a casein kinase II recognition site were shown to facilitate intracellular sorting of both furin and the mannose-6-phosphate receptor (Schafer et al., 1995;Voorhees et al., 1995;Mauxion et al., 1996). The functioning of sorting signals requires their direct (or possibly indirect) interaction with adaptor protein (AP) complexes, such as AP1, AP2, and AP3, which assemble with clathrin during vesicular budding (for review, see Kirchhausen et al., 1997;Odorizzi et al., 1998), or with arrestins, which function as adaptors for G-protein-coupled receptors (Ferguson et al., 1996;Goodman et al., 1996).Whereas t...

Phosphatidylinositol-4-Kinase Type II α Is a Component of Adaptor Protein-3-derived Vesicles

“…The kinase extensively colocalizes with AP-3 and ZnT3 and to a lower extent with AP-1. A-C to AЈ-CЈ series represent two optical planes taken every 0.75 m. Bar, 6 m. been described in the Golgi complex (Wang et al, 2003), synaptic vesicles (Guo et al, 2003), and endosomes (Balla et al, 2002), an organelle that in PC12 cell gives raise to microvesicles by AP-3-dependent mechanisms (Faundez et al, 1997;Shi et al, 1998;Blagoveshchenskaya et al, 1999;Salazar et al, 2004a,b). As a first step to determine the potential localization of PI4KII␣ to AP-3-derived vesicles, fractions from PC12 cell glycerol velocity gradients and isopycnic sucrose gradients leading to the ZnT3-enriched vesicle preparation ( Figure 1C) were probed with antibodies directed against PI4KII␣.…”

“…This drug blocks AP-1 (Robinson and Kreis, 1992) and AP-3 recruitment to membranes and robustly inhibits the formation of vesicles generated by AP-3-dependent mechanisms (Faundez et al, 1997;Shi et al, 1998;Blagoveshchenskaya et al, 1999;Salazar et al, 2004a,b). In contrast, it does not affect formation of AP-2-derived SLMVs (Faundez et al, 1997;Shi et al, 1998;Blagoveshchenskaya et al, 1999;Salazar et al, 2004a,b). As expected, the ZnT3 containing peak in glycerol velocity gradients almost disappeared after BFA treatment.…”

Cellugyrin and Synaptogyrin Facilitate Targeting of Synaptophysin to a Ubiquitous Synaptic Vesicle-sized Compartment in PC12 Cells

“…On the one hand, this model has been useful for elucidating SLMV targeting sequences for synaptic vesicle proteins, such as synaptobrevin and synaptotagmin (6,7). It is assumed that SLMV targeting sequences in PC12 cells would correspond to synaptic vesicle targeting sequences in neurons.…”

“…Thus PC12 cells have been used as an in vitro model for studying the biogenesis of SVs/SLMVs. Using this cell type, targeting signals have been identified in the amino acid sequence of the synaptic vesicle proteins VAMPII/ synaptobrevin (6) and synaptotagmin (7). These studies, however, have not identified a universal targeting signal.…”

Cellugyrin Induces Biogenesis of Synaptic-like Microvesicles in PC12 Cells

“…The group of nonsynaptic vesicle proteins localized in SLMVs can be further divided into the nonneuronal paralogs of synaptic vesicle proteins (cellubrevin, cellugyrin, and pantophysin) (14 -16) and those proteins that are unrelated to synaptic vesicle proteins (tyrosinase and P-selectin) (13,17). These studies, which examined targeting motifs, failed to identify a universal sequence applicable to all SLMV proteins.…”