A Two-Dimensional Electrophoresis Study of Phosphorylation and Dephosphorylation of Chromaffin Cell Proteins in Response to a Secretory Stimulus

Phosphorylated proteins of bovine chromaffin cells, radioactively labeled with [32P]orthophosphate, have been analyzed by two-dimensional polyacrylamide gel electrophoresis and autoradiography. Complex two-dimensional electrophoretograms were studied with the aid of computer-assisted image analysis (CAIA). A database map of 32P-labeled proteins was constructed; approximately 500 polypeptides have been detected, numbered, and characterized according to the intensity of labeling, molecular weight, and isoelectric point. The database was constructed from cells kept in resting conditions or stimulated with 59 mM K+ in 2.5 mM Ca2+ or in 0 Ca2+ solution. These manipulations caused statistically significant changes in the degree of phosphorylation of 20 proteins; they were classified as Ca2+-dependent substrates for the phosphorylation or dephosphorylation processes. These changes were also shown in cells stimulated in the presence of the Ca2+ channel activator Bay K 8644. New proteins that show as much as a fivefold increase in their phosphorylation state during cell stimulation have been located with this methodology, as well as many others that had not previously been detected with conventional methods. These experiments provide the first CAIA database of chromaffin cell phosphoproteins; the map constructed with these data will allow the location of specific phosphoproteins and serve as a reference for future ongoing studies. The database will continue to grow to identify more proteins and to facilitate the comparison of complex patterns obtained in different laboratories for normal and transformed pheochromocytoma PC12 cells.     

A complete energy balance from photons to new biomass reveals a light- and nutrient-dependent variability in the metabolic costs of carbon assimilation

The energy balance of Phaeodactylum tricornutum cells from photon to biomass have been analysed under nutrient-replete and N-limiting conditions in combination with fluctuating (FL) and non-fluctuating (SL) dynamic light. For this purpose, the amount of photons absorbed has been related to electrons transported by photosystem II, to gas exchange rates, and to the newly formed biomass differentially resolved into carbohydrates, proteins, and lipids measured by means of Fourier transform infrared (FTIR) spectroscopy. Under high nutrient conditions, the quantum efficiency of carbon-related biomass production (Phi(C)) and the metabolic costs of carbon (C) production were found to be strongly controlled by the light climate. Under N-limited conditions, the light climate was less important for the efficieny of primary production. Thus, the largest range of Phi(C) dependent on the nutrient status of the cells was observed under non-fluctuating light conditions which are comparable with stratified conditions in the natural environment. It is evident that N limitation induced pronounced changes in the composition of macromolecular compounds and, thus, influenced the degree of reduction of the biomass as well as the metabolic costs of C production. However, Phi(C) and the metabolic costs are not predictable from the photosynthesis rates. In consequence, the results clearly show that bio-optical methods as well as gas exchange measurements during the light phase can severely mismatch the true energy storage in the biomass especially under high nutrient in combination with non-fluctuating light conditions.     

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A,and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex

Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA_i. Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA_i in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA_i. Finally, we show that the C-terminal domain of eIF5 is responsible for the factor''s activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.