Rab11, a small GTPase associated with both constitutive and regulated secretory pathways in PC12 cells

Abstract

A specific polyclonal antibody was used to investigate the subcellular distribution of the small GTPase, rabl 1 p, in the neuroendocrine cell line, PC12. We took advantage of a previously described pulse-chase protocol based on sulfation [1] to examine the distribution of rabl 1 along the secretory pathway. Using the rabl 1 antiserum, but not serum depleted of rabl 1 antibodies, we were able to specifically immunoisolate markers of the constitutive and the regulated secretory pathway in the trans-Golgi network (TGN) as well as after their exit from this compartment (constitutive secretory vesicles, immature, and mature secretory granules). We therefore conclude that rabl lp is associated with the TGN and with TGN-derived vesicles of both the constitutive and the regulated secretory pathway in PC12 cells.

Rab11 Is Required for Trans-Golgi Network–to–Plasma Membrane Transport and a Preferential Target for GDP Dissociation Inhibitor

“…In this re-spect it is interesting to note that rab5, which has a high intrinsic GTPase activity and rapidly cycles between the GTP and GDP-bound form on membranes (Rybin et al, 1996), is relatively insensitive to release by GDI under our assay conditions (our unpublished results). In contrast, both rab7 and rab11 have significantly slower intrinsic GTPase activities (Urbé and Parton, 1995;Simon et al, 1996), yet both are sensitive to release from membranes by excess GDI (our unpublished results). Taken together, the data hint that the factors involved in stable membrane recruitment, including GDI displacement factor and guanine nucleotide exchange factor, may be the most critical in dictating the GDP-/GTP-bound ratio of an individual rab protein.…”

“…In nonpolarized cells rab11 was found associated with both the Golgi and the recycling en-dosome Ren et al, 1998). In polarized and regulated secretory cells rab11 has been localized to the Golgi as well as a variety of specialized membrane compartments (Urbé et al, 1993;Deretic et al, 1996;Goldenring et al, 1996Goldenring et al, , 1997Sheehan et al, 1996;Calhoun and Goldenring, 1997). The complex localization profiles of rab11 in diverse cell types have complicated the assessment of its precise function in intracellular membrane transport.…”

“…Along the exocytic pathway, endoplasmic reticulum (ER)-to-Golgi transport is regulated by two rab proteins, rab1 and rab2, and intra-Golgi transport depends on the action of rab6 (Plutner et al, 1991;Tisdale et al, 1992;Martinez et al, 1994). Transport from the trans-Golgi network (TGN) to the cell surface requires rab8 and possibly rab11 Urbé et al, 1993). Rab8 was shown to be important in the transport of vesicular stomatitis virus (VSV) G protein and Semliki Forest virus spike glycoproteins to the cell surface of polarized and nonpolarized cells, respectively (Ikonen et al, 1995;Peränen et al, 1996).…”

“…The evidence that mammalian rab11 might also play a role in exocytic membrane traffic is largely circumstantial and based solely on localization and cofractionation studies (Urbé et al, 1993;Deretic et al, 1996). The recent finding that the closest homologues of rab11 in yeast, Ypt31/32, are required for exit from the trans-Golgi suggested that this issue needed to be examined more directly (Jedd et al, 1997).…”

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

“…No detectable signal could be observed in the nuclear and PM (Figure 3A a, e, i, m). It also showed that there was a colocalization of rSac3 with concanavalin A (ConA), an ER marker ( Figure 3A a-d) [37], MCFD2, the ER-Golgi intermediate compartment marker (Figure 3A e-h) [35], WGA, a Golgi complex marker ( Figure 3A i-l) [38] and Rab11, a trans-Golgi network and recycling endosome marker ( Figure 3A m-p) [39,40]. No colocalization of rSac3 with the early endosome marker early endosomal antigen 1 (Supplementary information Figure S1D-F) [41], the late endosome marker mannose 6-phosphate receptor Figure S1G-I) [7] or the lipid fraft marker caveolin-1 [42] could be detected.…”