Subcellular localization and trafficking of phytolongins (non-SNARE longins) in the plant secretory pathway

Abstract

SNARE proteins are central elements of the machinery involved in membrane fusion of eukaryotic cells. In animals and plants, SNAREs have diversified to sustain a variety of specific functions. In animals, R-SNARE proteins called brevins have diversified; in contrast, in plants, the R-SNARE proteins named longins have diversified. Recently, a new subfamily of four longins named 'phytolongins' (Phyl) was discovered. One intriguing aspect of Phyl proteins is the lack of the typical SNARE motif, which is replaced by another domain termed the 'Phyl domain'. Phytolongins have a rather ubiquitous tissue expression in Arabidopsis but still await intracellular characterization. In this study, we found that the four phytolongins are distributed along the secretory pathway. While Phyl2.1 and Phyl2.2 are strictly located at the endoplasmic reticulum network, Phyl1.2 associates with the Golgi bodies, and Phyl1.1 locates mainly at the plasma membrane and partially in the Golgi bodies and post-Golgi compartments. Our results show that export of Phyl1.1 from the endoplasmic reticulum depends on the GTPase Sar1, the Sar1 guanine nucleotide exchange factor Sec12, and the SNAREs Sec22 and Memb11. In addition, we have identified the Y48F49 motif as being critical for the exit of Phyl1.1 from the endoplasmic reticulum. Our results provide the first characterization of the subcellular localization of the phytolongins, and we discuss their potential role in regulating the secretory pathway.

Keywords: ER export YF motif; longin domain; phytolongins; protein targeting; secretory pathway; subcellular localization..

Fig. 1.

Subcellular localization of the four phytolongins in tobacco leaf epidermal cells. (A, B) GFP-Phyl2.1 and GFP-Phyl2.2 appear to be localized in the ER network. (C) GFP-Phyl1.2 shows a typical co-labelling of the Golgi bodies as observed through its co-expression with the Golgi marker Erd2-YFP. (D, E) YFP-Phyl1.1 co-localized with the plasma membrane markers PMA4-YFP (D) and RFP-SYP121 (E).

Fig. 2.

Intracellular localization of the phytolongin Phyl1.1in tobacco leaf epidermal cells. (A, B) YFP-Phyl1.1 co-localized to some extent with the Golgi marker ST-RFP and the TGN marker RFP-SYP61. (C, D) YFP-Phyl1.1 also co-localized to some extent with the PVC marker RFP-VSR2 and the LPVC marker RFP-Rha1. E, Quantification of the co-localization of Phyl1.1 with the various markers, determined according to Gershlick et al. (2014). The far right panels in A–D show scatter plots derived from the correlation analysis.

Fig. 3.

Phyl1.1 is not recovered in DIM-enriched fractions and does not appear in the same pattern as remorin-like domains in situ. (A) Remorin (REM; a DIM marker) is enriched in fractions 1 and 2 (DIM, <10% of total proteins) and present in starting/solubilized membranes (fractions 3–9, >90% of total proteins). However, Phyl1.1 is not enriched in the DIM fractions and is mostly found in the starting/solubilized membranes. GFP-Phyl1.1-transformed leaves of 4-week-old tobacco plants were homogenized and centrifuged to produce a microsomal pellet. Triton X-100 lysis of membranes, separation into nine fractions on a sucrose gradient, and protein analysis by western blotting with antibodies to remorin and to GFP for revealing GFP-Phyl1.1 were performed as described in the Materials and methods. (B–M) When GFP-Phyl1.1 at the plasma membrane is analysed at the tangential plane by confocal microscopy (B–I), it behaves in exactly the same way as PMA4-GFP (L, M), which is not recovered in DIM, but not in the same way as GFP-Rem1.3 (J, K), which is a marker of DIM. (F–I) Images show slices from a z-stack.

Fig. 4.

Over-expression of Sar1 mutants and of Sec12 induces the redistribution of YFP-Phyl1.1 to the ER. (A, B) Expression of the mutant blocked forms of Sar1, Sar1-GDP and Sar1-GTP, inhibits transport of the protein YFP-Phyl1.1 to the PM and maintains it in the ER network. (C, D) Over-expression of Sec12 redistributes YFP-Phyl1.1 together with the co-expressed ST-RFP into the ER (D) compared with the control (C).

Fig. 5.

Over-expression of the SNAREs Sec22-CFP (A) and Memb11-CFP (B) redistributes YFP-Phyl1.1 from the PM into the ER.

Fig. 6.

Structural models for the Phyl1.1 and Phyl 1.2 longin domains. Opaque (top) and partially transparent (bottom, to highlight secondary structure) surface representations of the structural models for the LD of Phyl1.1 (left images) and Phyl1.2 (right images). The α1-β3 region is highlighted in yellow. The YF motif specific to Phyl1.1 and Phyl1.2 (red) is surface exposed, while the YF motif shared by all four phytolongins (blue) is buried in the LD core.

Fig. 7.

Trafficking motifs of Phyl1.1. Top: Amino acid sequences of Phyl1.1 and its mutated regions. Two putative ER export motifs, YF, are highlighted in blue; the TMD (24 amino acids; highlighted in red) was shortened, deleting the six amino acids in italic text. (A) Normal localization of YFP-Phyl1.1 at the PM. (B) The mutated YFP-Phyl1.1Y48G/F49G is retained in the ER network. (C) The mutated YFP-Phyl1.1TMD is localized at the PM, indicating that shortening the TMD did not affect its transport to the PM. (D, E) Co-expression of YFP-Phyl1.1Y48G/F49G with the ER marker RFP-HDEL and the Golgi marker ST-RFP. (F, G) Retention of YFP-Phyl1.1(GF)(Y48G/F49) and YFP-Phyl1.1(FF)(Y48F/F49) at the beginning of the secretory pathway (mostly in the ER) by co-expression with the respective ER marker RFP-HDEL. (H) Phyl1.2(GG)(Y50G/F51G) forms large aggregates co-localizing with the ER network.