Members of a mammalian SNARE complex interact in the endoplasmic reticulum in vivo and are found in COPI vesicles.

Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an intermediate compartment. Here, we report that the mammalian soluble N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.

Live cell FRET microscopy: homo- and heterodimerization of two human peroxisomal ABC transporters, the adrenoleukodystrophy protein (ALDP, ABCD1) and PMP70 (ABCD3).

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half-ATP-binding cassette (ABC) transporters in the mammalian peroxisome membrane. Mutations in the gene encoding ALDP result in a devastating neurodegenerative disorder, X-linked adrenoleukodystrophy (X-ALD) that is associated with elevated levels of very long chain fatty acids because of impaired peroxisomal beta-oxidation. The interactions of peroxisomal ABC transporters, their role in the peroxisomal membrane, and their functions in disease pathogenesis are poorly understood. Studies on ABC transporters revealed that half-transporters have to dimerize to gain functionality. So far, conflicting observations are described for ALDP. By the use of in vitro methods (yeast two-hybrid and immunoprecipitation assays) on the one hand, it was shown that ALDP can form homodimers as well as heterodimers with PMP70 and ALDR, while on the other hand, it was demonstrated that ALDP and PMP70 exclusively homodimerize. To circumvent the problems of artificial interactions due to biochemical sample preparation in vitro, we investigated protein-protein interaction of ALDP in its physiological environment by FRET microscopy in intact living cells. The statistical relevance of FRET data was determined in two different ways using probability distribution shift analysis and Kolmogorov-Smirnov statistics. We demonstrate in vivo that ALDP and PMP70 form homodimers as well as ALDP/PMP70 heterodimers where ALDP homodimers predominate. Using C-terminal deletion constructs of ALDP, we demonstrate that the last 87 C-terminal amino acids harbor the most important protein domain mediating these interactions, and that the N-terminal transmembrane region of ALDP has an additional stabilization effect on ALDP homodimers. Loss of ALDP homo- or heterodimerization is highly relevant for understanding the disease mechanisms of X-ALD.

Structural elucidation of the PDI-related chaperone Wind with the help of mutants.

The structures of the PDI-related protein Wind (with a C-terminal His(6) tag) and the mutants Y53S, Y53F and Y55K have been determined and compared with the wild-type structure with the His(6) tag at the N-terminus. All five structures show the same mode of dimerization, showing that this was not an artefact introduced by the nearby N-terminal His(6) tag and suggesting that this dimer may also be the biologically active form. Although the mutants Y53S and Y55K completely abrogate transport of the protein Pipe (which appears to be the primary function of Wind in the cell), only subtle differences can be seen in the putative Pipe-binding region. The Pipe binding in the active forms appears to involve hydrophobic interactions between aromatic systems, whereas the inactive mutants may be able to bind more strongly with the help of hydrogen bonds, which could disturb the delicate equilibrium required for effective Pipe transport.

Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells.

Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB(10463) between Leu(543) and Gly(544) and the naturally occurring variant TcdB(8864) between Leu(544) and Gly(545). The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB(10463)) and 62 761 Da (TcdB(8864))] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB(10463)) and 206 245 Da (TcdB(8864))] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

Mapping of a substrate binding site in the protein disulfide isomerase-related chaperone wind based on protein function and crystal structure.

The protein disulfide isomerase (PDI)-related protein Wind is essential in Drosophila melanogaster, and is required for correct targeting of Pipe, an essential Golgi transmembrane 2-O-sulfotransferase. Apart from a thioredoxin fold domain present in all PDI proteins, Wind also has a unique C-terminal D-domain found only in PDI-D proteins. Here, we show that Pipe processing requires dimeric Wind, which interacts directly with the soluble domain of Pipe in vitro, and we map an essential substrate binding site in Wind to the vicinity of an exposed cluster of tyrosines within the thioredoxin fold domain. In vitro, binding occurs to multiple sites within the Pipe polypeptide and shows specificity for two consecutive aromatic residues. A second site in Wind, formed by a cluster of residues within the D-domain, is likewise required for substrate processing. This domain, expressed separately, impairs Pipe processing by the full-length Wind protein, indicating competitive binding to substrate. Our data represent the most accurate map of a peptide binding site in a PDI-related protein available to date and directly show peptide specificity for a naturally occurring substrate.

4Pi-microscopy of the Golgi apparatus in live mammalian cells.

We report the applicability of 4Pi-microscopy to live mammalian cells. Controlled interference of the counterpropagating wavefronts is possible despite the slight variations in cellular refractive index. Superresolved 3D-fluorescence imaging is exemplified with the first representation of the Golgi apparatus in a live cell at approximately 100 nm resolution.

Crystal structure and functional analysis of Drosophila Wind, a protein-disulfide isomerase-related protein.

In the developing Drosophila melanogaster embryo, dorsal-ventral patterning displays an absolute requirement for the product of the essential windbeutel gene, Wind. In homozygous windbeutel mutant flies, dorsal-ventral patterning fails to initiate because of the failure of the Golgi-resident proteoglycan-modifying protein, Pipe, to exit the endoplasmic reticulum, and this leads to the death of the embryo. Here, we describe the three-dimensional structure of Wind at 1.9-A resolution and identify a candidate surface for interaction with Pipe. This represents the first crystal structure of a eukaryotic protein-disulfide isomerase-related protein of the endoplasmic reticulum to be described. The dimeric protein is composed of an N-terminal thioredoxin domain and a C-terminal alpha-helical domain unique to protein-disulfide isomerase D proteins. Although Wind carries a CXXC motif that is partially surface accessible, this motif is redox inactive, and the cysteines are not required for the targeting of Pipe to the Golgi. However, both domains are required for targeting Pipe to the Golgi, and, although the mouse homologue ERp28 cannot replace the function of Wind, exchange of the Wind D-domain with that of ERp28 allows for efficient Golgi transport of Pipe.

Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy.

Fluorescence resonance energy transfer (FRET) resolved by multifocal multiphoton microscopy (MMM) was successfully used to measure transport phenomena in living cells. We expressed different pairs of CFP-/YFP-fusion proteins involved in retrograde Golgi-to-ER transport to analyze sorting of the occupied KDEL-receptor into retrograde transport vesicles triggered by application of the external cholera toxin mutant CTXK63. FRET observed as a sensitized emission of the acceptor was confirmed by acceptor photobleaching and the dequenching of the donor was measured. FRET-MMM data obtained from single cells were compared with bulk cell experiments employing spectrofluorimetry. The importance of controlling the degree of overexpression of CFP-/YFP-fusion proteins for FRET analysis is stressed in this article. Using MMM we showed for the first time that FRET can be measured across the Golgi membrane. Finally, FRET-MMM records performed continuously over 2 h allowed to analyze intracellular retrograde transport and sorting events and to discuss these mechanisms on a single cell level.

Differential expression of receptors for Shiga and Cholera toxin is regulated by the cell cycle.

Cholera and Shiga toxin bind to the cell surface via glycolipid receptors GM1 and Gb3, respectively. Surprisingly, the majority of Vero cells from a non-synchronized population bind either Cholera or Shiga toxin but not both toxins. The hypothesis that the differential expression of toxin receptors is regulated by the cell cycle was tested. We find that Cholera toxin binds preferentially in G0/G1, with little binding through S-phase to telophase, whereas Shiga toxin binds maximally through G2 to telophase but does not bind during G0/G1 and S-phase. The changes result from the corresponding changes in Gb3 and GM1 synthesis, not from variations of receptor transport to the cell surface. The changes do not reflect competition of Gb3 and GM1 synthesis for lactosylceramide. Cells as diverse as Vero cells, PC12 cells and astrocytes show the same cell-cycle-dependent regulation of glycosphingolipid receptors, suggesting that this novel phenomenon is based on a conserved regulatory mechanism.