Biochemistry and Molecular Pharmacology
Biochemistry and Molecular Biology
First Thesis Advisor
Carlos B. Hirschberg Ph.D.
Endoplasmic Reticulum, Golgi Apparatus, Nucleotides
In mammals, newly synthesized proteins destined for secretion are translocated cotranslationally into the lumen of the Endoplasmic Reticulum (ER). Once inside, these nascent polypeptide chains are bound by a lumenal ER protein called BiP (Immunoglobulin Binding Protein) or Grp 78 (Glucose Regulated Protein 78). It is hypothesized that this binding is necessary to protect the nascent chains until they are properly folded or assembled with other subunits. When the proteins are folded and assembled, they are released from BiP by a process that is dependent on ATP hydrolysis. Since ATP is synthesized mainly in the mitochondria, we hypothesized that there must be an ATP transporter in the ER which would allow the protein mediated transport of ATP from the cytosol into the ER lumen. We studied the transport of ATP in vitro and found that ATP enters the lumen of the ER in a saturable manner with a Kmapp~3μM. ATP transport is dependent on time, protein, and vesicle integrity, it is also inhibited by the general anion transport inhibitor, 4,4' diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). We also found that the transport was inhibited by membrane impermeable protein modifying agents such as N-ethlymaleamide (NEM) and Pronase when added to intact ER vesicles. These results suggest that the transport is mediated by a protein with an active cytoplasmic face. Using monoclonal and polyclonal antibodies to BiP and Grp94 (another resident ER protein) and U.V. crosslinking, we demonstrated that after transport of ATPα32P into intact vesicles, radiolabeled BiP and Grp94 could be immunoprecipitated. We also found that labeling of lumenal proteins with ATP is dependent on the transport of ATP. Finally using ATP labeled with 35S, we concluded that BiP was able to bind intact ATP and we confirmed earlier work that BiP was thiophosphorylated while Grp94 is not.
The second area of study involves processes that occur further along the secretory pathway in the Golgi apparatus. It was known from previous work that the nucleotide sugar substrates necessary for the synthesis of the linkage region, UDP-xylose (UDP-Xyl), UDP-galactose (UDP-Gal) and UDP-glucuronic acid (UDP-GlcA) were transported into the Golgi apparatus from the cytosol via protein mediated transporters. In order to eventually purify one of these transporter proteins, we wanted to reconstitute their activities. We were able to reconstitute the activities that exhibited kinetic parameters and inhibitor sensitivities very similar to those seen in intact Golgi vesicles. In the case of UDP-xylose it was necessary to prepare the liposomes using endogenous Golgi lipids in order to get transport activity similar to that seen in the intact Golgi vesicles. This suggested a specific lipid requirement for the UDP-xylose transporter. These transporters seem to be antiporters, whereby the nucleotide sugar enters the lumen of the Golgi coupled to the equimolar exit of the corresponding nucleoside monophosphate (Hirschberg, C.B. and Snider, M.D. 1987). We also showed that we could reproduce the hypothesized antiporter system in the reconstituted proteoliposomes by preloading the proteoliposomes with the putative antiporter molecule UMP.
The rationale for developing the reconstituted system is eventually to use this system to purify one of these nucleotide sugar translocators. In the last set of studies, I have shown that this reconstituted system can be used to monitor the purification of the UDP-galactose translocator. Using column chromatography we were able to purify this membrane translocator protein 45,000 fold from a rat liver homogenate.
Clairmont, CA. Transport of Nucleotide Derivatives into Endoplasmic Reticulum and Golgiapparatus Derived Vesicles: a Dissertation. (1993). University of Massachusetts Medical School. GSBS Dissertations and Theses. Paper 77. DOI: 10.13028/w4aw-7473. https://escholarship.umassmed.edu/gsbs_diss/77
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