The intercellular and intracellular distribution of the movement protein (MP) of the Ob tobamovirus was examined in infected leaf tissues using an infectious clone of Ob in which the MP gene was translationally fused to the gene encoding the green fluorescent protein (GFP) of Aequorea victoria. In leaves of Nicotiana tabacum and N. benthamiana, the modified virus caused fluorescent infection sites that were visible as expanding rings. Microscopy of epidermal cells revealed subcellular patterns of accumulation of the MP:GFP fusion protein which differed depending upon the radial position of the cells within the fluorescent ring. Punctate, highly localized fluorescence was associated with cell walls of all of the epidermal cells within the infection site, and apparently represents association of the fusion protein with plasmodesmata; furthermore, fluorescence was retained in cell walls purified from infected leaves. Within the brightest region of the fluorescent ring, the MP:GFP was observed in irregularly shaped inclusions in the cortical regions of infected cells. Fluorescent filamentous structures presumed to represent association of MP:GFP with microtubules were observed, but were distributed differently within the infection sites on the two hosts. Within cells containing filaments, a number of fluorescent bodies, some apparently streaming in cytoplasmic strands, were also observed. The significance of these observations is discussed in relation to MP accumulation, targeting to plasmodesmata, and degradation.

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg͞ml without aggregation and displayed Ϸ70% ␣-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative ␣-helical transmembrane domains and a proteasesensitive carboxyl terminus.T he plus-sense, 6.4-kb single-stranded (ss) RNA genome of tobacco mosaic virus (TMV) encodes a 17.5-kDa coat protein, a 30-kDa movement protein (MP), and proteins of 126 kDa and 183 kDa that function in virus replication (1). The MP is essential for cell-cell spread of infection (2, 3).Many proteins, including several plant virus movement proteins (3), are associated with intracellular and intercellular channels that permit passage of water, ions, metabolites, and signaling molecules into and between cells and cell compartments. Prokaryotic examples include porins (4), potassium channels (5), and aquaporins (6). Eukaryotic examples include aquaporins (7), gap junctions (8), translocation channels of the endoplasmic reticulum (ER; ref. 9), nuclear pore complexes (10), ryanodine receptors (11), and the acetylcholine receptor (12). In higher plants, the channels that mediate intercellular communication are termed plasmodesmata (3). Plasmodesmata allow passive transport of proteins of at least 50 kDa in young tobacco leaf tissues, but the size exclusion limit decreases as these tissues mature (13). TMV infection results in a temporary increase in the size exclusion limit of plasmodesmata from Ϸ0.4 kDa to Ϸ20 kDa in mature leaf epidermis and mesophyll tissues (14). Although the MP is required for this dramatic and transient increase in intercellular permeability, the mechanisms responsible are unclear (3).Many viruses replicate in association with ER membranes, and some viruses associate with the cytoskeleton of the host (15-22). MP behaves as an intrinsic membrane protein, promotes the formation of ER aggregates, and probably facilitates establishment of TMV replication complexes that contain viral RNA, replicase, and MP (16,20,[22][23][24]. Many recombinant viral MPs expressed in Escherchia coli bind ss nucleic acids in vitro without nucleotide sequence specificity (25-29). Thus, it was proposed that the MP functions as an intracellular and intercellular carrier of TMV RNA, at least in part by association with the cytoskeleton and ER membranes (15)(16)(17)22).Recombinant viral MPs typically form insoluble inclusion bodies (25,...

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.

SummaryTo identify and map functionally important regions of the tobacco mosaic virus movement protein, deletions of three amino acids were introduced at intervals of 10 amino acids throughout the protein. Mutations located between amino acids 1 and 160 abolished the capacity of the protein to transport virus from cell to cell, while some of the mutations in the C-terminal third of the protein permitted function. Despite extensive tests, no examples were found of intermolecular complementation between mutants, suggesting that function requires each movement protein molecule to be fully competent. Many of the mutants were fused to green fluorescent protein, and their subcellular localizations were determined by fluorescence microscopy in infected plants and protoplasts. Most mutants lost the ability to accumulate in one or more of the multiple subcellular sites targeted by wild-type movement protein, suggesting that specific functional domains were disrupted. The order in which accumulation at subcellular sites occurs during infection does not represent a targeting pathway. Association of the movement protein with microtubules or with plasmodesmata can occur in the absence of other associations. The region of the protein around amino acids 9-11 may be involved in targeting the protein to cortical bodies (probably associated with the endoplasmic reticulum) and to plasmodesmata. The region around residues 49-51 may be involved in co-alignment of the protein with microtubules. The region around residues 88-101 appears to play a role in targeting to both the cortical bodies and microtubules. Thus, the movement protein contains independently functional domains.