Metabolic behavior of cell surface biotinylated proteins

Abstract

The turnover of proteins on the surface of cultured mammalian cells was measured by a new approach. Reactive free amino or sulfhydryl groups on surface-accessible proteins were derivatized with biotinyl reagents and the proteins solubilized from culture dishes with detergent. Solubilized, biotinylated proteins were than adsorbed onto streptavidin-agarose, released with sodium dodecyl sulfate and mercaptoethanol, and separated on polyacrylamide gels. Biotin-.epsilon.-aminocaproic acid N-hydroxysuccinimide ester (BNHS) or N-biotinoyl-N''-(maleimidohexanoyl)hydrazine (BM) were the derivatizing agents. Only 10-12 bands were adsorbed onto streptavidin-agarose from underivatized cells or from derivatized cells treated with free avidin at 4.degree. C. Two-dimensional isoelectric focusing-sodium dodecyl sulfate gel electrophoresis resolved > 100 BNHS-derivatized proteins and > 40 BM-derivatized proteins. There appeared to be little overlap between the two groups of derivatized proteins. Short-term pulse-chase studies showed an accumulation of label into both groups of biotinylated proteins up until 1-2 h of chase and a rapid decrease over the next 1-5 h. Delayed appearance of labeled protein at the cell surface was attributed to transit time from site of synthesis. The unexpected and unexplained rapid disappearance of pulse-labeled proteins from the cell surface was invariant for all two-dimensionally resolved proteins and was sensitive to temperature reduction to 18.degree. C. Long-term pulse-chase experiments beginning 4-8 h after the initiation of chase showed the disappearance of derivatized proteins to be a simple first-order process having a half-life of 115 h in the case of BNHS-derivatized proteins and 30 h in the case of BM-derivatized proteins. Pulse-chase analysis of biotinylated proteins by double labeling with [3H]methionine and [35S]methionine and their resolution on two-dimensional polyacrylamide gels allowed the rate of disappearance for a number of plasma membrane proteins to be examined. Four BNHS-reactive proteins examined by this approach between 8 and 48 h of chase demonstrated similar slow turnover, mirroring the long half-life determined for bulk BNHS-derivatized proteins. The turnover rates for eight BM-derivatized proteins were more heterogeneous than that for BNHS-derivatized proteins. Four of the latter exhibited T1/2 .ltoreq. 22 h.