Membrane insertion and biogenesis of the Turnip crinkle virus p9 movement protein

Abstract

Plant viral infection and spread depends on the successful introduction of a virus into a cell of a compatible host, followed by replication and cell-to-cell transport. The movement proteins (MPs) p8 and p9 of Turnip crinkle virus are required for cell-to-cell movement of the virus. We have examined the membrane association of p9 and found that it is an integral membrane protein with a defined topology in the endoplasmic reticulum (ER) membrane. Furthermore, we have used a site-specific photo-cross-linking strategy to study the membrane integration of the protein at the initial stages of its biosynthetic process. This process is cotranslational and proceeds through the signal recognition particle and the translocon complex.

FIG. 1.

TCV p9 MP is an integral membrane protein. Segregation of [35S]Met-labeled p9 into membranous and soluble fractions (untreated, lanes 1 and 2) and after alkaline wash (sodium carbonate buffer, lanes 3 and 4) or urea treatment (lanes 5 and 6). P and S denote pellet and supernatant, respectively. For Triton X-114 partitioning experiments, OP and AP refer to organic and aqueous phases, respectively (lanes 7 and 8). A.E., alkaline extraction.

FIG. 2.

Insertion of TCV p9 hydrophobic region (HR) 1 and 2 into microsomal membranes. (Top) Schematic representation of the leader peptidase (Lep) construct used to report insertion into the ER membrane of p9 HR1 and HR2. The HR under study is inserted into the P2 domain of Lep, flanked by two artificial glycosylation acceptor sites (G1 and G2). Recognition of the HR by the translocon machinery as a TM domain locates only G1 on the luminal side of the ER membrane, preventing G2 glycosylation. The Lep chimera will be doubly glycosylated when the HR being tested is translocated into the lumens of the microsomes. (Middle) In vitro translation in the presence of membranes of the different Lep constructs. Control HRs were used to verify sequence translocation (lane 1) and membrane integration (lane 2) (clones 67 and 68 in reference [44], a kind gift from G. von Heijne's lab). Constructs containing HR1 (residues 3 to 20), HR2 (residues 38 to 57), or HR2 with Leu49 replaced by Asp (lanes 3, 4 and 5, respectively) were transcribed and translated in the presence of membranes. Bands of nonglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively. The HR sequence in each construct is shown at the bottom.

FIG. 3.

Insertion and topology of p9-50P2. (A) In vitro translation of wild-type (wt) p9 (lanes 1 to 5) and the L49D mutant (lanes 6 to 8) when fused to the first 50 amino acids of the P2 domain from Lep, in the presence (+) and absence (−) of microsomal membranes (MMs) and proteinase K (PK). Bands of nonglycosylated protein are indicated by a white dot; glycosylated proteins are indicated by a black dot. The arrowhead identifies undigested protein after PK treatment (with either 0.2 or 0.4 mg/ml). (B) Triton X-114 partitioning of Lep, PNRSV p32 MP (a previously reported nonintegral membrane protein [33]), and p9-50P2. OP and AP refer to organic and aqueous phases, respectively.

FIG. 4.

TCV p9 MP topology. (Top) Schematic representation of the constructs used in the study of p9 topology. The locations of acceptor sites are indicated with a white or a black dot indicating nonglycosylation and glycosylation, respectively. (Bottom) In vitro translation in the presence (+) and absence (−) of microsomal membranes (MMs) and endoglycosidase H (Endo H; a glycan-removing enzyme) of samples encoding engineered glycosylation sites either in the soluble C-t domain (N77) (lanes 1 to 3) or in the middle of HR2 (N45) (lanes 3 to 6). Translation of N77 and N45 mutants fused to the first 50 amino acids of the P2 domain from Lep were also included (lanes 7 to 9). Bands of nonglycosylated protein are indicated by a white dot; singly and doubly glycosylated proteins are indicated by one and two black dots, respectively.

FIG. 5.

TCV p9 integrates into the membrane cotranslationally and interacts with SRP. (A) p9 (harboring an engineered glycosylation site at the C-t, position 77) was translated in either the absence (lanes 1 and 3) or the presence (lanes 2 and 4) of microsomal membranes. In lane 3, microsomal membranes were added posttranslationally (after 1 h; Post) and incubation was continued for another 1 h. In lane 4, the p9 construct was translated in the presence of cotranslationally added membranes (Co) and treated later with Endo H. (B) Photo-cross-linking of TCV p9 to SRP. Structural organization of p9 (top). A single photoreactive probe was incorporated by positioning an amber stop codon at position 12. RNCs containing 70-residue radioactive nascent chains were prepared in the presence of unmodified Lys-tRNA^amb or photoreactive ɛANB-Lys-tRNA^amb, as indicated. Only the sample in lane 4 was supplemented with exogenous SRP. aas, amino acids.

FIG. 6.

Photo-cross-linking of TCV p9 to Sec61α and TRAM. After photolysis in the presence of membranes, an aliquot from each RNC (70 and 110 residues) was removed and directly analyzed by SDS-PAGE to detect and normalize the total radioactive translation products (labeled as totals). The remaining samples were split for IPs with Sec61α (lanes 2 and 5) and TRAM (lanes 3 and 6) antisera.