Note added in proofWhile this work was under review, the Asna-1 protein was independently identified as a cytosolic ATPase that can promote the membrane insertion of TA proteins (Stefanovic and Hegde, 2007). We therefore conclude that the ATPdependent integration of TA proteins at the ER is most probably complex and multifaceted.

Signal recognition particle (SRP) plays a central role in the delivery of classical secretory and membrane proteins to the endoplasmic reticulum (ER). All nascent chains studied to date dissociate from SRP once released from the ribosome, thereby supporting a strictly cotranslational mode of action for eukaryotic SRP. We now report a novel post-translational function for SRP in the targeting of tail-anchored (TA) proteins to the ER. TA proteins possess a hydrophobic membrane insertion sequence at their C-terminus such that it can only emerge from the ribosome after translation is terminated. We show that SRP can associate post-translationally with this type of ER-targeting signal, and deliver newly synthesised TA proteins to the ER membrane by a pathway dependent upon GTP and the SRP receptor. We find that dependency upon this SRP-dependent route is precursor specific, and propose a unifying model to describe the biogenesis of TA proteins in vivo.

SUMMARYAuxin is a major growth hormone in plants, and recent studies have elucidated many of the molecular mechanisms underlying its action, including transport, perception and signal transduction. However, major gaps remain in our knowledge of auxin biosynthetic control, partly due to the complexity and probable redundancy of multiple pathways that involve the YUCCA family of flavin-dependent mono-oxygenases. This study reveals the differential localization of YUCCA4 alternative splice variants to the endoplasmic reticulum and the cytosol, which depends on tissue-specific splicing. One isoform is restricted to flowers, and is anchored to the cytosolic face of the endoplasmic reticulum membrane via a hydrophobic C-terminal transmembrane domain. The other isoform is present in all tissues and is distributed throughout the cytosol. These findings are consistent with previous observations of yucca4 phenotypes in flowers, and suggest a role for intracellular compartmentation in auxin biosynthesis.

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N-and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-P-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-P-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

The endoplasmic reticulum (ER) is a major site of protein synthesis and its inside, or lumen, is a major site of protein folding. The lumen of the ER contains many folding factors and molecular chaperones, which facilitate protein folding by increasing both the rate and the efficiency of this process. Amongst the many ER folding factors, there are three components that specifically modulate the folding glycoproteins bearing N-linked carbohydrate side chains. These components are calnexin, calreticulin and ERp57, and this review focuses on the molecular basis for their capacity to influence glycoprotein folding. ß