Quantitative ER <-> Golgi transport kinetics and protein separation upon Golgi exit revealed by vesicular integral membrane protein 36 dynamics in live cells

To quantitatively investigate the trafficking of the transmembrane lectin V IP36 and its relation to cargo-containing transport carriers (TCs), we anal yzed a C-terminal fluorescent-protein (FP) fusion, VIP36-SP-FP. When expres sed at moderate levels, VIP36-SP-FP localized to the endoplasmic reticulum, Golgi apparatus, and intermediate transport structures, and colocalized wi th epitope-tagged VIP36. Temperature shift and pharmacological experiments indicated VIP-36-SP-FP recycled in the early secretory pathway, exhibiting trafficking representative of a class of transmembrane cargo receptors, inc luding the closely related lectin ERGIC53. VIP36-SP-FP trafficking structur es comprised tubules and globular elements, which translocated in a saltato ry manner. Simultaneous visualization of anterograde secretory cargo and VI P36-SP-FP indicated that the globular structures were pre-Golgi carriers, a nd that VIP36-SP-FP segregated from cargo within the Golgi and was not incl uded in post-Golgi TCs. Organelle-specific bleach experiments directly meas ured the exchange of VIP36-SP-FP between the Golgi and endoplasmic reticulu m. (ER). Fitting a two-compartment model to the recovery data predicted fir st order rate constants of 1.22 +/- 0.44%/min for ER --> Golgi, and 7.68 +/ - 1.94%/min for Golgi --> ER transport, revealing a half-time of 113 +/- 70 min for leaving the ER and 1.67 +/- 0.45 min for leaving the Golgi, and ac counting for the measured steady-state distribution of VIP36-SP-FP (13% Gol gi/87% ER). Perturbing transport with AlF4- treatment altered VIP36-SP-GFP distribution and changed the rate constants. The parameters of the model su ggest that relatively small differences in the first order rate constants, perhaps manifested in subtle differences in the tendency to enter distinct TCs, result in large differences in the steady-state localization of secret ory components.