SummaryWe have established a procedure for isolating native peroxisomal membrane protein complexes from cultured human cells. Protein-A-tagged peroxin 14 (PEX14), a central component of the peroxisomal protein translocation machinery was genomically expressed in Flp-In-293 cells and purified from digitonin-solubilized membranes. Size-exclusion chromatography revealed the existence of distinct multimeric PEX14 assemblies at the peroxisomal membrane. Using mass spectrometric analysis, almost all known human peroxins involved in protein import were identified as constituents of the PEX14 complexes. Unexpectedly, tubulin was discovered to be the major PEX14-associated protein, and direct binding of the proteins was demonstrated. Accordingly, peroxisomal remnants in PEX14-deficient cells have lost their ability to move along microtubules. In vivo and in vitro analyses indicate that the physical binding to tubulin is mediated by the conserved N-terminal domain of PEX14. Thus, human PEX14 is a multi-tasking protein that not only facilitates peroxisomal protein import but is also required for peroxisome motility by serving as membrane anchor for microtubules.

Background: Cryptophytes like Guillardia theta utilize soluble phycobiliproteins for light-harvesting. Results: Guillardia theta adopted phycoerythrobilin biosynthesis from cyanobacteria, and the phycobiliprotein lyase GtCPES provides structural requirements for transfer of this chromophore to a specific cysteine residue of the apophycobiliprotein. Conclusion: Phycobiliprotein synthesis in Guillardia theta combines proven and novel components. Significance: Results provide a better understanding of the evolution and function of unusual phycobiliproteins in cryptophytes.

The protein targeting signal recognition particle (SRP) pathway in chloroplasts of higher plants has undergone dramatic evolutionary changes. It disposed of its RNA, which is an essential SRP component in bacteria, and uses a unique chloroplastspecific protein cpSRP43. Nevertheless, homologs of the conserved SRP54 and the SRP receptor, FtsY, are present in higher plant chloroplasts. In this study, we analyzed the phylogenetic distribution of SRP components in photosynthetic organisms to elucidate the evolution of the SRP system. We identified conserved plastid SRP RNAs within all nonspermatophyte land plant lineages and in all chlorophyte branches. Furthermore, we show the simultaneous presence of cpSRP43 in these organisms. The function of this novel SRP system was biochemically and structurally characterized in the moss Physcomitrella patens. We show that P. patens chloroplast SRP (cpSRP) RNA binds cpSRP54 but has lost the ability to significantly stimulate the GTPase cycle of SRP54 and FtsY. Furthermore, the crystal structure at 1.8-Å resolution and the nucleotide specificity of P. patens cpFtsY was determined and compared with bacterial FtsY and higher plant chloroplast FtsY. Our data lead to the view that the P. patens cpSRP system occupies an intermediate position in the evolution from bacterial-type SRP to higher plant-type cpSRP system.

In contrast to the majority of cyanobacteria, the unicellular marine cyanobacterium Prochlorococcus marinus MED4 uses an intrinsic divinyl-chlorophyll-dependent light-harvesting system for photosynthesis. Despite the absence of phycobilisomes, this high-light adapted strain possesses ␤-phycoerythrin (CpeB), an S-type lyase (CpeS), and enzymes for the biosynthesis of phycoerythrobilin (PEB) and phycocyanobilin. Of all linear tetrapyrroles synthesized by Prochlorococcus including their 3Z-and 3E-isomers, CpeS binds both isomers of PEB and its biosynthetic precursor 15,16-dihydrobiliverdin (DHBV). However, dimerization of CpeS is independent of bilins, which are tightly bound in a complex at a ratio of 1:1. Although bilin binding by CpeS is fast, transfer to CpeB is rather slow. CpeS is able to attach 3E-PEB and 3Z-PEB to dimeric CpeB but not DHBV. CpeS transfer of 3Z-PEB exclusively yields correctly bound ␤Cys 82 -PEB, whereas ␤Cys 82 -DHBV is a side product of 3E-PEB transfer. Spontaneous 3E-and 3Z-PEB addition to CpeB is faulty, and products are in both cases ␤Cys 82 -DHBV and likely a PEB bound at ␤Cys 82 in a non-native configuration. Our data indicate that CpeS is specific for 3Z-PEB transfer to ␤Cys 82 of phycoerythrin and essential for the correct configuration of the attachment product.

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA-blocked mutant with all possible monofluoro 3-aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.