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BiP (Kar2 in yeast) is an essential Hsp70 chaperone and master regulator of endoplasmic reticulum (ER) function. BiP's activity is regulated by its intrinsic ATPase activity that can be stimulated by two different nucleotide exchange factors, Sil1 and Lhs1. Both Sil1 and Lhs1 are glycoproteins, but how N-glycosylation regulates their function is not known. Here, we show that N-glycosylation of Sil1, but not of Lhs1, is diminished upon reductive stress. N-glycosylation of Sil1 is predominantly Ost3-dependent and requires a functional Ost3 CxxC thioredoxin motif. N-glycosylation of Lhs1 is largely Ost3-independent and independent of the CxxC motif. Unglycosylated Sil1 is not only functional but is more effective at rescuing loss of Lhs1 activity than N-glycosylated Sil1. Furthermore, substitution of the redox active cysteine pair C52 and C57 in the N terminus of Sil1 results in the Doa10-dependent ERAD of this mutant protein. We propose that reductive stress in the ER inhibits the Ost3-dependent N-glycosylation of Sil1,which regulates specific BiP functions appropriate to the needs of the ER under reductive stress. [ABSTRACT FROM AUTHOR]

Examines the dual function of vanadate as a transition state analogue and as a photoactive chemical. Definition of the adenosine triphosphate-binding cassette (ABC) superfamily; Basis for the cooperativity observed in the nucleotide-binding domains of ABC transporters; Use of vanadate photocleavage to identify residues in close contact to nucleotide.

Acute lymphoblastic leukemia (ALL) is the major pediatric cancer. At diagnosis, the developmental timing of mutations contributing critically to clonal diversification and selection can be buried in the leukemia's covert natural history. Concordance of ALL in monozygotic, monochorionic twins is a consequence of intraplacental spread of an initiated preleukemic clone. Studying monozygotic twins with ALL provides a unique means of uncovering the timeline of mutations contributing to clonal evolution, pre- and postnatally. We sequenced the whole genomes of leukemic cells from two twin pairs with ALL to comprehensively characterize acquired somatic mutations in ALL, elucidating the developmental timing of all genetic lesions. Shared, prenatal, coding-region single-nucleotide variants were limited to the putative initiating lesions. All other nonsynonymous single-nucleotide variants were distinct between tumors and, therefore, secondary and postnatal. These changes occurred in a background of noncoding mutational changes that were almost entirely discordant in twin pairs and likely passenger mutations acquired during leukemic cell proliferation. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the movement of substances across the membrane; conformational changes clearly play an important role in the transporter mechanism. Previously, we have shown that a dimer of Malk, the ATPase subunit of the maltose transporter from Escherichia coli, undergoes a tweezers-like motion in a transport cycle. The MalK monomer consists of an N-terminal nucleotide binding domain and a Ceterminal regulatory domain. The two nucleotide- binding domains in a dimer are either open or closed, depending on whether ATP is present, while the regulatory domains maintain contacts to hold the dimer together. In this work, the structure of MalK in a posthydrolysis state is presented, obtained by cocrystallizing MalK with ATP-Mg2+. ATP was hydrolyzed in the crystallization drop, and ADP-Mg2+ was found in the resulting crystal structure. In contrast to the ATP-bound form where two ATP molecules are buried in a closed interface between the nucleotide-binding domains, the two nucleotide-binding domains of the ADP-bound form are open, indicating that ADP, unlike ATP, cannot stabilize the closed form. This conclusion is further supported by oligomerization studies of MalK in solution. At low protein concentrations, ATP promotes dimerization of MalK, whereas ADP does not. The structures of dimeric MalK in the nucleotide-free, ATP-bound, and ADP-bound forms provide a framework for understanding the nature of the conformational changes that occur in an ATP-binding cassette transporter hydrolysis cycle, as well as how conformational changes in MalK are coupled to solute transport. [ABSTRACT FROM AUTHOR]

ATP-binding cassette (ABC) transporters are powered by a nucleotide-binding domain dimer that opens and closes during cycles of ATP hydrolysis. These domains consist of a RecA-like subdomain and an α-helical subdomain that is specific to the family. Many studies on isolated domains suggest that the helical subdomain ro- tates toward the RecA-like subdomain in response to AlP binding, moving the family signature motif into a favorable position to interact with the nucleotide across the dimer interface. Moreover, the transmembrane domains are docked into a cleft at the interface between these subdomains. suggesting a putative role of the rotation in interdomain communication. Electron paramagnetic resonance spectroscopy was used to study the dynamics of this rotation in the intact Escherichia coil maltose transporter MaIFGK2+. This importer requires a periplasmic maltose-binding protein (MBP) that activates AlP hydrolysis by promoting the closure of the cassette dimer (MalK2). Whereas this rotation occurred during the transport cycle, it required not only trinucleotide, but also MBP, suggesting it is part of a global conformational change in the transporter. Interaction of AMP-PNP-Mg2+ and a MBP that is locked in a closed conformation induced a transition from open MaIK2 to semiopen MalK2 without significant subdomain rotation. Inward rotation of the helical subdomain and complete closure of MaIK2 therefore appear to be coupled to the reorientation of transmembrane helices and the opening of MBP. events that promote transfer of maltose into the transporter. After ATP hydrolysis, the helical subdomain rotates out as MaIK2 opens, resetting the transporter in an inward-facing conformation. [ABSTRACT FROM AUTHOR]

The maltose transporter MalFGK2 of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MaIFGK2 and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MaIFGK2 during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP. as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell. [ABSTRACT FROM AUTHOR]