Domains of tobacco mosaic virus movement protein essential for its membrane association

Abstract

A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains. INTRODUCTIONCell-to-cell movement of plant viruses requires one or more virus-encoded proteins that enable infectious entities to traverse the plasmodesmata (Pd). Pd are structural channels that provide cytoplasmic and endoplasmic reticulum (ER) continuity between adjacent cells (Staehelin, 1997;Oparka et al., 1999;Lazarowitz & Beachy, 1999). Infection by Tobacco mosaic virus (TMV) leads to production of a virusencoded 30 kDa movement protein (MP) that is required for cell-to-cell movement of the complex that contains viral RNA. MP induces a transient modification of Pd, resulting in an increase in size exclusion limit (SEL) of the Pd from 0?5 to over 10 kDa (Deom et al., 1987;Oparka et al., 1997).Virus replication complexes (VRCs) of many different viruses are associated with one or more cellular organelles, including lysosomal or endosomal membranes (Froshauer et al., 1988; Kujala et al., 2001), peroxisomes (Bleve-Zacheo et al., 1997), mitochondrial outer membranes (Miller et al., 2001), chloroplast envelope (Prod'homme et al., 2003) or vacuolar membranes (van der Heijden et al., 2001; Hasegawa et al., 2003). In the case of TMV, the VRCs assemble on ER membranes. VCRs contain viral RNA, viral replicase and MP plus many unidentified host proteins, including elements of the cell cytoskeleton such as microtubules and actin filaments (Mas & Beachy, 1999;Lazarowitz & Beachy, 1999;Beachy & Heinlein, 2000;Asurmendi et al., 2004). More recently, real-time imaging of TMV infection that monitored the accumulation of MPgreen fluorescent protein (GFP) fusion protein provided evidence that TMV infection spreads from cell to cell as intact VRCs (Kawakami et al., 2004). Because of the intimate association of MP with ER and its role in forming VRCs on ER, it is important to characterize the association of MP with ER. The hypothesis that MP contains two transmembrane domains that might anchor the protein (Berna, 1995) was supported by recent studies using purified recombinant MP (Brill et al., 2000(Brill ...

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily

“…In the same manner, a (conservative) substitution of the equivalent Leu with Val or Ile resulted in improved TMV movement (Toth et al, 2002;Kawakami et al, 2003). In contrast, substitutions of the equivalent b1 residue abolished virus movement in TRV (this study), and in CPMV, Chinese wheat mosaic virus (CWMV) and Prunus necrotic ringspot virus (PNRSV), as well as tubule formation in CPMV (Bertens et al, 2000, Martínez-Gil et al, 2009Andika et al, 2013), whilst deletion of part of the b1 region prevented TMV movement (Fujiki et al, 2006).…”

“…On the one hand, a series of experiments on TMV 30K concluded that it was most probably an integral membrane protein with two a-helical transmembrane segments (although a tight association with membranes without transmembrane domains could not be excluded; Brill et al, 2000;Fujiki et al, 2006). The two transmembrane segments correspond to the regions predicted as strands b1 and b6 in the study of Melcher (2000), which is the nomenclature followed in the present study.…”

Membrane Insertion and Biogenesis of the Turnip Crinkle Virus p9 Movement Protein

“…MPs belong to different protein families with unique functional and structural characteristics. The most studied MP is p30 from the Tobacco mosaic virus, a 30-kDa RNA-binding protein (4) with two putative transmembrane (TM) segments (2) that has so far been considered an integral membrane protein (13,42). At an early stage of infection, p30 associates with the ER network (18,59).…”

Control of Tobacco mosaic virus Movement Protein Fate by CELL-DIVISION-CYCLE Protein48

“…The MP is a hydrophobic protein that behaves like a membrane-integral or tightly membraneassociated protein in differential fractionation experiments and contains two predicted transmembrane domains Brill et al, 2000Brill et al, , 2004 involved in ER association (Fujiki et al, 2006). The association with microtubules depends on MP amino acids 1 to 213 required for MP function (Kahn et al, 1998;Boyko et al, 2000bBoyko et al, , 2000cBoyko et al, , 2002Kotlizky et al, 2001).…”