Demonstration in Yeast of the Function of BP-80, a Putative Plant Vacuolar Sorting Receptor

Abstract

BP-80, later renamed VSR PS-1 , is a putative receptor involved in sorting proteins such as proaleurain to the lytic vacuole, with its N-terminal domain recognizing the vacuolar sorting determinant. Although all VSR PS-1 characteristics and in vitro binding properties described so far favored its receptor function, this function remained to be demonstrated. Here, we used green fluorescent protein (GFP) as a reporter in a yeast mutant strain defective for its own vacuolar receptor, Vps10p. By expressing VSR PS-1 together with GFP fused to the vacuolar sorting determinant of petunia proaleurain, we were able to efficiently redirect the reporter to the yeast vacuole. VSR PS-1 is ineffective on GFP either alone or when fused with another type of plant vacuolar sorting determinant from a chitinase. The plant VSR PS-1 therefore interacts specifically with the proaleurain vacuolar sorting determinant in vivo, and this interaction leads to the transport of the reporter protein through the yeast secretory pathway to the vacuole. This finding demonstrates VSR PS-1 receptor function but also emphasizes the differences in the spectrum of ligands between Vps10p and its plant equivalent. INTRODUCTIONThe plant secretory system is far less easy to study than its yeast counterpart, mainly due to the lack of an equivalent mutant collection. Plant and yeast cells, in opposition to mammalian cells, do not use the mannose 6-phosphatebased lysosomal sorting tag (Kornfeld, 1992); instead, the sorting information is carried by a peptidic sequence. In yeast, the two vacuolar proteins carboxypeptidase Y (CPY; Johnson et al., 1987) and proteinase A (Klionsky and Emr, 1989) both are produced with an N-terminal propeptide responsible for the vacuolar location of the mature protein. Almost 50 mutants defective for vacuolar protein sorting ( vps ) have allowed the identification of proteins involved in the yeast vacuolar sorting pathway. One of these proteins, Vps10p, plays a central role because it is the vacuolar receptor for CPY (Marcusson et al., 1994). Vps10p also is able to send foreign or malfolded proteins to the vacuole for degradation in a process that is believed to be independent of any sorting signal (Hong et al., 1996).In plants, two main types of vacuolar sorting determinants (VSDs) have been well studied and are believed to correspond to two separate vacuolar sorting pathways. The first type could be defined as a sequence-specific VSD (ssVSD) and has been well studied for barley aleurain (Holwerda et al., 1992) and sweet potato sporamin A (Matsuoka and Nakamura, 1991). For these two examples, the VSD is located within an N-terminal propeptide and contains a conserved tetrapeptide NPIR, the Ile being essential (Matsuoka and Nakamura, 1999). The position of this category of VSD seems to be less important than its sequence specificity, as shown with sporamin A (Koide et al., 1997). The second type of VSD is found in C-terminal propeptides (Ct-VSD); it is rather short with no obvious sequence conservation but needs to be accessibl...

Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting

“…The vacuolar accumulation of msGFP resembles previous observations that GFP variants were targeted to the yeast vacuole [21,35]. Intriguingly, vacuolar targeting of GFP was shown to depend on the sorting receptor Vps10 [21].…”

“…Budding yeasts contain a vacuolar sorting receptor called Vps10 that recognizes the carboxypeptidase Y (CPY) precursor as well as certain misfolded proteins [15][16][17][18][19][20]. When msGFP was targeted to the secretory pathway in S. cerevisiae, some of the msGFP molecules reached the vacuole in a Vps10-dependent manner, confirming a report that the quality control function of Vps10 extends to folded GFP [21]. Building on a previous functional dissection of Vps10 [15], we found that deletion of a single domain of Vps10 prevented diversion of msGFP to the vacuole without causing missorting of CPY.…”

“…Intriguingly, vacuolar targeting of GFP was shown to depend on the sorting receptor Vps10 [21]. We therefore engineered a vps10Δ strain, and then repeated the fluorescence microscopy analysis with preOst1-msGFP ( Figure 5A).…”

Identification of Multivesicular Bodies as Prevacuolar Compartments inNicotiana tabacumBY-2 Cells[W]

“…However, BP-80 also is known to bind to the C-terminal propeptide of 2S albumin, which lacks the NPIR motif (Kirsch et al, 1996). BP-80 is a member of a family of membrane proteins termed vacuolar sorting receptors (VSRs; Paris et al, 1997;Shimada et al, 1997;Hadlington and Denecke 2000;Paris and Neuhaus, 2002), whose function has been confirmed both in vitro (Cao et al, 2000) and in vivo using a yeast expression system (Humair et al, 2001). Targeting determinants also have been identified for proteins sorted into the PSV (Frigerio et al, 1998;Neuhaus and Rogers, 1998).…”

A Slow Maturation of a Cysteine Protease with a Granulin Domain in the Vacuoles of Senescing Arabidopsis Leaves

“…The barley (Hordeum vulgare) aleurain (Rogers et al, 1985) and sweet potato (Ipomoea batatas) sporamin (Murakami et al, 1986) have a vacuolar targeting signal, Asn-Pro-IleArg (NPIR), in their NTPPs Nakamura, 1991, 1999;Chrispeels and Raikhel, 1992). The NPIR sequence is recognized by a vacuolar sorting receptor such as pea (Pisum sativum) BP-80 (Paris et al, 1997;Humair et al, 2001), pumpkin (Cucurbita maxima) PV72 (Shimada et al, 1997), and Arabidopsis AtELP (Ahmed et al, 2000). On the other hand, NTPPs of human cathepsin B (P07858) and cathepsin S (M90696) function as an autoinhibitory domain that covers the active site within the molecule (Turk et al, 1996;Maubach et al, 1997).…”