Identification of Sorting Motifs of AtβFruct4 for Trafficking from the ER to the Vacuole Through the Golgi and PVC

Abstract

Although much is known about the molecular mechanisms involved in transporting soluble proteins to the central vacuole, the mechanisms governing the trafficking of membrane proteins remain largely unknown. In this study, we investigated the mechanism involved in targeting the membrane protein, AtβFructosidase 4 (AtβFruct4), to the central vacuole in protoplasts. AtβFruct4 as a green fluorescent protein (GFP) fusion protein was transported as a membrane protein during transit from the endoplasmic reticulum (ER) through the Golgi apparatus and the prevacuolar compartment (PVC). The N-terminal cytosolic domain of AtβFruct4 was sufficient for transport from the ER to the central vacuole and contained sequence motifs required for trafficking. The sequence motifs, LL and PI, were found to be critical for ER exit, while the EEE and LCPYTRL sequence motifs played roles in trafficking primarily from the trans Golgi network (TGN) to the PVC and from the PVC to the central vacuole, respectively. In addition, actin filaments and AtRabF2a, a Rab GTPase, played critical roles in vacuolar trafficking at the TGN and PVC, respectively. On the basis of these results, we propose that the vacuolar trafficking of AtβFruct4 depends on multiple sequence motifs located at the N-terminal cytoplasmic domain that function as exit and/or sorting signals in different stages during the trafficking process.

Arabidopsis μ-adaptin subunit AP1M of adaptor protein complex 1 mediates late secretory and vacuolar traffic and is required for growth

“…To identify trafficking pathways involving AP1M2, we examined the effect of the ap1m2-1 mutation on various post-Golgi trafficking pathways. Soluble cargo proteins sporamin and Arabidopsis aleurain-like protease (AALP) and the membrane cargo protein AtβFructosidase4 were used as GFP-fusion proteins to analyze vacuolar trafficking in ap1m2-1 protoplasts (36,37). Unlike the situation in WT, these proteins were not processed in the ap1m2-1 mutant, as seen by the increase in intensity of the precursor band, and this defect was suppressed in protoplasts from ap1m2-1 plants expressing AP1M2:myc (Fig.…”

Dual-targeting of Arabidopsis DMP1 isoforms to the tonoplast and the plasma membrane

“…After synthesis at the ER membrane, cargo membrane proteins are incorporated into coat protein complex II (COPII) vesicles [52] upon interaction between coat proteins and ER export signals before delivery to the Golgi [53, 54]. Various export signals have been described in yeast, mammals and plants, including diacidic (D/E-X-D/E), dihydrophobic (ISI), diaromatic motifs (FF, YY and FY) [35, 37, 38, 51, 55, 56] and the motif PI [36]. Impaired or altered recognition of export signals due to mutations or masking by fusion moieties may result in partial or total retention of the protein within the ER membrane.…”

Trans-Golgi Network-Located AP1 Gamma Adaptins Mediate Dileucine Motif-Directed Vacuolar Targeting in Arabidopsis

“…However, it remains elusive if the roles of TMD length in targeting would be applied to multiple spanning membrane proteins. The demonstration of various sorting signals in multiple TMD proteins such as SCAMP1 further indicated the complexity of the targeting mechanism of proteins with multiple TMDs in plant cells (Cai et al, 2011;Jung et al, 2011).…”

“…The C terminus of tonoplast two-pore K+ channels has been proved to contain targeting information for different types of vacuoles both in rice (Oryza sativa) and Arabidopsis thaliana (Isayenkov et al, 2011;Maîtrejean et al, 2011). Another study with Arabidopsis b-Fructosidase 4 has identified EEE and LCPYTRL sequence motifs as sorting signals on the way from the TGN to the central vacuole (Jung et al, 2011). Similar to cases in mammalian cells and yeast, a conserved dileucine motif is also involved in the targeting of tonoplast membrane proteins in plant cells, as recorded for several proteins like the monosaccharide transporter ERD SIX-LIKE1 (ESL1), the molybdate transporter2 (MOT2), two pore channel1 (TPC1), peptide transporter1 (PTR1), and inositol transporter1 (INT1) (Yamada et al, 2010;Gasber et al, 2011;Komarova et al, 2012;Larisch et al, 2012;Wolfenstetter et al, 2012).…”