Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex.

BACKGROUND: Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. RESULTS: Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM). CONCLUSION: Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.

Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway.

Tomato bushy stunt virus (TBSV), a positive-strand RNA virus, causes extensive inward vesiculations of the peroxisomal boundary membrane and formation of peroxisomal multivesicular bodies (pMVBs). Although pMVBs are known to contain protein components of the viral membrane-bound RNA replication complex, the mechanisms of protein targeting to peroxisomal membranes and participation in pMVB biogenesis are not well understood. We show that the TBSV 33-kD replication protein (p33), expressed on its own, targets initially from the cytosol to peroxisomes, causing their progressive aggregation and eventually the formation of peroxisomal ghosts. These altered peroxisomes are distinct from pMVBs; they lack internal vesicles and are surrounded by novel cytosolic vesicles that contain p33 and appear to be derived from evaginations of the peroxisomal boundary membrane. Concomitant with these changes in peroxisomes, p33 and resident peroxisomal membrane proteins are relocalized to the peroxisomal endoplasmic reticulum (pER) subdomain. This sorting of p33 is disrupted by the coexpression of a dominant-negative mutant of ADP-ribosylation factor1, implicating coatomer in vesicle formation at peroxisomes. Mutational analysis of p33 revealed that its intracellular sorting is also mediated by several targeting signals, including three peroxisomal targeting elements that function cooperatively, plus a pER targeting signal resembling an Arg-based motif responsible for vesicle-mediated retrieval of escaped ER membrane proteins from the Golgi. These results provide insight into virus-induced intracellular rearrangements and reveal a peroxisome-to-pER sorting pathway, raising new mechanistic questions regarding the biogenesis of peroxisomes in plants.

Membrane-bound fatty acid desaturases are inserted co-translationally into the ER and contain different ER retrieval motifs at their carboxy termini.

Fatty acid desaturases (FADs) play a prominent role in plant lipid metabolism and are located in various subcellular compartments, including the endoplasmic reticulum (ER). To investigate the biogenesis of ER-localized membrane-bound FADs, we characterized the mechanisms responsible for insertion of Arabidopsis FAD2 and Brassica FAD3 into ER membranes and determined the molecular signals that maintain their ER residency. Using in vitro transcription/translation reactions with ER-derived microsomes, we show that both FAD2 and FAD3 are efficiently integrated into membranes by a co-translational, translocon-mediated pathway. We also demonstrate that while the C-terminus of FAD3 (-KSKIN) contains a functional prototypic dilysine ER retrieval motif, FAD2 contains a novel C-terminal aromatic amino acid-containing sequence (-YNNKL) that is both necessary and sufficient for maintaining localization in the ER. Co-expression of a membrane-bound reporter protein containing the FAD2 C-terminus with a dominant-negative mutant of ADP-ribosylation factor (Arf)1 abolished transient localization of the reporter protein in the Golgi, indicating that the FAD2 peptide signal acts as an ER retrieval motif. Mutational analysis of the FAD2 ER retrieval signal revealed a sequence-specific motif consisting of Phi-X-X-K/R/D/E-Phi-COOH, where -Phi- are large hydrophobic amino acid residues. Interestingly, this aromatic motif was present in a variety of other known and putative ER membrane proteins, including cytochrome P450 and the peroxisomal biogenesis factor Pex10p. Taken together, these data describe the insertion and retrieval mechanisms of FADs and define a new ER localization signal in plants that is responsible for the retrieval of escaped membrane proteins back to the ER.