Protein transport from the endoplasmic reticulum (ER) to the Golgi requires the action of the Rab1 GTPase. Rab1 cycles between a GDP and a GTP-bound form, and its activity is mediated by the interaction of Rab1-GTP with different proteins. To study the dynamics of Rab1 we have used time lapse microscopy of living cells (HeLa) transfected with GFP-Rab1. The GFP-Rab1 localized predominantly to the Golgi and to numerous peripheral punctate structures. The GFP-Rab1 labeled punctate structures are largely immobile. The majority shows limited movements of less than 2 mm in diameter during time periods of 5 minutes. Only occasionally, longer range, randomly directed movements up to 10 mm were observed. The binding and release kinetic of GFP-Rab1 to and from Golgi membranes and peripheral punctate structures was analyzed by fluorescence recovery after photobleaching (FRAP). Bleached GFP-Rab1 shows rapidly recovery in both compartments with a half time (t1/2) of 80 seconds. Taken together our data suggest that: 1) Rab1 recruited to peripheral structures does not move towards the Golgi; 2) Golgi association of Rab1 occurs only as a result of its exchange between Golgi membranes and cytosol; 3) Dissociation rate of Rab1 from Golgi or punctate structures is similar, suggesting that the same mechanism may regulate Rab1 dynamics in both membrane compartments.
