A Vacuolar Sorting Domain May Also Influence the Way in Which Proteins Leave the Endoplasmic Reticulum

Abstract

Protein sorting to plant vacuoles is known to be dependent on a considerable variety of protein motifs recognized by a family of sorting receptors. This can involve either traffic from the endoplasmic reticulum (ER) through the Golgi apparatus or direct ER-to-vacuole transport. Barley aspartic protease (Phytepsin) was shown previously to reach the vacuole via trafficking through the Golgi apparatus. Here we show that Phytepsin normally exits the ER in a COPII-mediated manner, because the Phytepsin precursor accumulates in the ER upon specific inhibition of the formation of COPII vesicles in vivo. Phytepsin differs from its yeast and mammalian counterparts by the presence of a saposin-like plant-specific insert (PSI). Deletion of this domain comprising 104 amino acids causes efficient secretion of the truncated molecule (Phytepsin ⌬ PSI) without affecting the enzymatic activity of the enzyme. Interestingly, deletion of the PSI also changes the way in which Phytepsin exits the ER. Inhibition of COPII vesicle formation causes accumulation of the Phytepsin precursor in the ER but has no effect on the secretion of Phytepsin ⌬ PSI. This suggests either that vacuolar sorting commences at the ER export step and involves recruitment into COPII vesicles or that the PSI domain carries two signals, one for COPII-dependent export from the ER and one for vacuolar delivery from the Golgi. The relevance of these observations with respect to the bulk flow model of secretory protein synthesis is discussed. INTRODUCTIONPlant cells contain at least two functionally distinct vacuolar compartments: the central lytic vacuole, which is related to the mammalian lysosome, and the so-called storage vacuoles, which appear to be unique to the plant kingdom (Marty, 1999). Whereas the lytic vacuole is typical for vegetative cells, storage vacuoles are found mostly in reserve tissues of seeds. Exceptions to this are the vegetative storage vacuoles that are formed during stress conditions and that could be related to a neutral vacuolar compartment recently discovered in tobacco protoplasts (Di Sansebastiano et al., 1998). It was first believed that the various vacuolar compartments share a common origin but change appearance and contents according to physiological conditions or tissue type. However, the simultaneous presence of storage vacuoles and lytic vacuoles within the same cell, as determined using specific membrane markers (Hoh et al., 1995;Paris et al., 1996;Jauh et al., 1998), argues against this notion. In addition, the great variety of vacuolar sorting signals described in plants (Matsuoka and Neuhaus, 1999) also suggests the possibility that the various types of vacuoles have different origins and are supported by different protein transport pathways (Chrispeels and Herman, 2000).The first evidence for distinct transport routes arose from the use of pharmacological agents that exhibit a differential effect on the vacuolar sorting of a variety of cargo molecules (Gomez and Chrispeels, 1993;Matsuoka et al., 1995). Furthermore, the to...

The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

“…Vacuolar sorting signals are grouped into three categories: sequencespecific signals, C-terminal signals, and physical structure signals (Vitale and Hinz, 2005). Three vacuolar proteins have been reported to possess physical structure vacuolar sorting signals (Saalbach et al, 1991;von Schaewen and Chrispeels, 1993;Tö rmä kangas et al, 2001). One of these, barley aspartic protease (phytepsin), was shown to reach the vacuoles through the Golgi apparatus in a coat protein complex (COP)II-mediated manner.…”

Sorting and Anterograde Trafficking at the Golgi Apparatus: Figure 1.

“…The saposin-like plant-specific insert present in barley and soybean (Glycine max) aspartic proteinases may also function as a vacuolar sorting signal (Tormakangas et al, 2001;Simões and Faro, 2004). Additional evidence is needed to confirm the role of the saposin-like plantspecific insert in vacuolar sorting at the Golgi complex.…”

Cell biology of the plant Golgi apparatus

“…However, it is clear that plants possess functional genes encoding the majority of the COPII machinery, as reported in yeast and mammals (Bar-Peled & Raikhel, 1997;Andreeva et al ., 1998b;Movafeghi et al ., 1999;Phillipson et al ., 2001). Biochemical studies and in vivo imaging using fluorescent protein technology have shown that over expression of Sec12, the Sar1p guanine nucleotide exchange factor (GEF) inhibits export out of the ER (Phillipson et al ., 2001;Törmäkanagas et al ., 2001;daSilva et al ., 2004), presumably by the titration of Sar1p away from putative ER exit sites. The requirement of the GTPase Sar1p for ER export has been directly demonstrated by the expression of nonfunctional mutant forms of the protein, which resulted in accumulation GFP tagged proteins in the ER which was interpreted as reflecting a block in ER export (Andreeva et al ., 2000;Takeuchi et al ., 2000;daSilva et al ., 2004).…”

“…Proteins such as the legume globulins and legumins destined for storage vacuoles (Hillmer et al, 2001), characterised by various combinations of α, δ, and γ-TIPs ( Jiang & Rogers, 2003) are transported in noncoated Golgi-derived vesicles, which have sometimes been termed 'dense vesicles' due to the osmiophyllic nature of their contents (Hohl et al, 1996;Hinz & Herman, 2003). Classic electron microscopy and immunogold labelling has shown that sorting into these vesicles can take place as early as the cis-Golgi (see Regulation of ER to Golgi transport).…”