The formation of ubiquitin rich condensates triggers recruitment of the ATG9A lipid transfer complex to initiate basal autophagy.

Autophagy is an essential cellular recycling process that maintains protein and organelle homeostasis. ATG9A vesicle recruitment is a critical early step in autophagy to initiate autophagosome biogenesis. The mechanisms of ATG9A vesicle recruitment are best understood in the context of starvation-induced non-selective autophagy, whereas less is known about the signals driving ATG9A vesicle recruitment to autophagy initiation sites in the absence of nutrient stress. Here we demonstrate that loss of ATG9A or the lipid transfer protein ATG2 leads to the accumulation of phosphorylated p62 aggregates in the context of basal autophagy. Furthermore, we show that p62 degradation requires the lipid scramblase activity of ATG9A. Lastly, we present evidence that poly-ubiquitin is an essential signal that recruits ATG9A and mediates autophagy foci assembly in nutrient replete cells. Together, our data support a ubiquitin-driven model of ATG9A recruitment and autophagosome formation during basal autophagy.

Identification of the cellulose-binding domain of the cellulosome subunit S1 from Clostridium thermocellum YS.

The 3' region of a gene designated cipB, which shows strong homology with cipA that encodes the cellulosome SL subunit of Clostridium thermocellum ATCC 27405, was isolated from a gene library of C. thermocellum strain YS. The truncated S1 protein encoded by the cipB derivative bound tightly to cellulose. The cellulose-binding domain in this polypeptide consisted of a C-terminal proximal 167 residue sequence which showed complete identity with residues 337-503 of mature SL from C. thermocellum strain ATCC 27405. The cellulose-binding domain interacted with both crystalline and amorphous cellulose, but not with xylan.

The role of conserved tryptophan residues in the interaction of a bacterial cellulose binding domain with its ligand.

The five conserved tryptophan residues in the cellulose binding domain of xylanase A from Pseudomonas fluorescens subsp. cellulosa were replaced with alanine and phenylalanine. The mutated domains were fused to mature alkaline phosphatase, and the capacity of the hybrid proteins to bind cellulose was assessed. Alanine substitution of the tryptophan residues, in general, resulted in a significant decrease in the capacity of the cellulose binding domains to bind cellulose. Mutant domains containing phenylalanine substitution retained some affinity for cellulose. The C-terminal proximal tryptophan did not play an important role in ligand binding, while Trp13, Trp34 and Trp38 were essential for the cellulose binding domain to retain cellulose binding capacity. Data presented in this study suggest major differences in the mechanism of cellulose attachment between Pseudomonas and Cellulomonas cellulose binding domains.

Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains.

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

Evidence for a general role for high-affinity non-catalytic cellulose binding domains in microbial plant cell wall hydrolases.

Cellulases expressed by Cellulomonas fimi consist of a catalytic domain and a discrete non-catalytic cellulose-binding domain (CBD). To establish whether CBDs are common features of plant cell-wall hydrolases from C. fimi, the molecular architecture of xylanase D (XYLD) from this bacterium was investigated. The gene encoding XYLD, designated xynD, consisted of an open reading frame of 1936 bp encoding a protein of M(r) 68,000. The deduced primary sequence of XYLD was confirmed by the size (64 kDa) and N-terminal sequence of the purified recombinant xylanase. Biochemical analysis of the purified enzyme revealed that XYLD is an endoacting xylanase which displays no detectable activity against polysaccharides other than xylan. The predicted primary structure of XYLD comprised an N-terminal signal peptide followed by a 190-residue domain that exhibited significant homology to Family-G xylanases. Truncated derivatives of xynD, encoding the N-terminal 193 amino acids of mature XYLD directed the synthesis of a functional xylanase, confirming that the 190-residue N-terminal sequence constitutes the catalytic domain. The remainder of the enzyme consisted of two approximately 90-residue domains, which exhibited extensive homology with each other, and limited sequence identity with CBDs from other polysaccharide hydrolases. Between the two putative CBDs is a 197-amino-acid sequence that exhibits substantial homology with Rhizobium NodB proteins. The four discrete domains in XYLD were separated by either threonine/proline-or novel glycine-rich linker regions. Although full-length XYLD adsorbed to cellulose, truncated derivatives of the enzyme lacking the C-terminal CBD hydrolysed xylan but did not bind to cellulose.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleotide sequence of the Ruminococcus albus SY3 endoglucanase genes celA and celB.

The complete nucleotide sequences of Ruminococcus albus genes celA and celB coding for endoglucanase A (EGA) and endoglucanase B (EGB), respectively, have been determined. The celA structural gene consists of an open reading frame of 1095 bp. Confirmation of the nucleotide sequence was obtained by comparing the predicted amino acid sequence with that derived by N-terminal analysis of purified EGA. The celB structural gene consists of an open reading frame of 1227 bp; 7 bp upstream of the translational start codon of celB is a typical gram-positive Shine-Dalgarno sequence. The deduced N-terminal region of EGB conforms to the general pattern for the signal peptides of secreted prokaryotic proteins. The complete celB gene, cloned into pUC vectors, caused lethality in Escherichia coli. In contrast, celA cloned in pUC18, under the control of lacZp, directed high-level synthesis of EGA in E. coli JM83. EGA in cell-free extract, purified to near homogeneity by ion-exchange chromatography, had a Mr of 44.5 kDa. Gene deletion and subcloning studies with celA revealed that EGA hydrolysed both CMC and xylan, and did not contain discrete functional domains. EGA and EGB showed considerable homology with each other, in addition to exhibiting similarity with Eg1 (R. albus), EGE (Clostridium thermocellum) and End (Butyrivibrio fibrisolvens).

Xylanase B and an arabinofuranosidase from Pseudomonas fluorescens subsp. cellulosa contain identical cellulose-binding domains and are encoded by adjacent genes.

The complete nucleotide sequence of the Pseudomonas fluorescens subsp. cellulosa xynB gene, encoding an endo-beta-1,4-xylanase (xylanase B; XYLB) has been determined. The structural gene consists of an open reading frame (ORF) of 1775 bp coding for a protein of Mr 61,000. A second ORF (xynC) of 1712 bp, which starts 148 bp downstream of xynB, encodes a protein, designated xylanase C (XYLC), of Mr 59,000. XYLB hydrolyses oat spelt xylan to xylobiose and xylose, whereas XYLC releases only arabinose from the same substrate. Thus XYLB is a typical xylanase and XYLC is an arabinofuranosidase. Both enzymes bind to crystalline cellulose (Avicel), but not to xylan. The nucleotide sequences between residues 114 and 931 of xynB and xynC were identical, as were amino acid residues 39-311 of XYLB and XYLC. This conserved sequence is reiterated elsewhere in the P. fluorescens subsp. cellulosa genome. Truncated derivatives of XYLB and XYLC, in which the conserved sequence had been deleted, retained catalytic activity, but did not exhibit cellulose binding. A hybrid gene in which the 5' end of xynC, encoding residues 1-110 of XYLC, was fused to the Escherichia coli pho A' gene (encodes mature alkaline phosphatase) directed the synthesis of a fusion protein which exhibited alkaline phosphatase activity and bound to cellulose.

Pregnancy after autotransplantation of the Fallopian tube in the ewe.

Vascularised transplantation of the Fallopian tube is a technically feasible operation, but its functional result remains questionable. This article documents a pregnancy after vascularised contralateral autotransplantation of the oviduct in a ewe.

Completed pregnancy following vascularized heterotopic autotransplantation of the Fallopian tube in the ewe.

Transplantation of the Fallopian tube is a technically feasible operation. Previous authors have reported pregnancies following turbo-ovarian transplants, but there appears to be no record of gestation following transplantation of the isolated oviduct. This communication illustrates the method that we have used to perform Fallopian tube transplants in the ewe and documents our first full-term pregnancy following this operation. It provides further evidence of the technical feasibility of Fallopian tube transplants.