Kinesin and dynamin are required for post-Golgi transport of a plasma-membrane protein

Abstract

n higher eukaryotes, secretory and plasma-membrane proteins are transported from the endoplasmic reticulum (ER) to a central Golgi complex and subsequently packaged into membrane-bound carriers for delivery to the cell surface. The long-distance transport of post-Golgi organelles to the tips of axons or to developing hyphal extensions in Neurospora crassa shows an absolute requirement for microtubules and microtubule-associated motors 1,2 . In contrast, microtubule disruption only moderately attenuates Golgi-toplasma-membrane transport in fibroblasts and randomizes surface delivery of select proteins in epithelial cells 3,4 . The observed preservation of biosynthetic transport after microtubule disruption is probably due to the extensive fragmentation and redistribution of Golgi mini-stacks to regions immediately adjacent to both the ER and the plasma membrane 3,5,6 . Here we have designed experiments to test the hypothesis that when the characteristic central localization of the Golgi is preserved, microtubules, kinesin and the GTPase dynamin are essential for post-Golgi trafficking. We ruled out a pharmacological approach to tackling this problem because we found that microtubule antagonists caused dispersal of the Golgi complex before complete microtubule disassembly occurred (see Supplementary Information). Instead, we microinjected functionblocking anti-kinesin antibodies HD and SUK-4 or cDNAs encoding a dominant-negative form of dynamin into cells expressing a green fluorescent protein (GFP)-tagged apical-membrane protein, p75. We show that kinesin and dynamin are required for different stages of post-Golgi transport.During a 2.5-h transport block at 20 °C, newly synthesized p75-GFP translocated from the ER to a juxtanuclear region (Fig. 1a, control, 0 min) and co-localized with Golgi/trans-Golgi network (TGN) markers (Fig. 1b). Within 4 h after shifting to the permissive temperature for transport, 32 °C, 81% of p75-GFP translocated from the Golgi to the plasma membrane (Fig. 1a, control, 240 min). The emptying rate of p75-GFP from the Golgi correlated with its arrival at the cell surface, as determined by immunocytochemical analysis of p75 in injected cells (data not shown) and by pulsechase, surface-biotinylation assays of p75 or p75-GFP in stable MDCK transfectants (see Supplementary Information). Normal trafficking of p75 was unaffected by the GFP tag: microinjected p75-GFP was selectively delivered to the apical membrane of confluent, polarized MDCK cells (data not shown). Figure 1 Inhibition of kinesin activity or expression of D2K44E inhibits release of p75-GFP from the Golgi/TGN after release from a block at 20 °C. a, Cells were microinjected with p75-GFP alone (control; n = 19) or with p75-GFP and HD (n = 13), SUK-4 (n = 8), wild-type dynamin-2 cDNA (n = 9) or cDNA encoding a dominantnegative dynamin-2, D2K44E (n = 15). Exit of p75-GFP from the Golgi/TGN was monitored by time-lapse imaging after shifting to 32 °C. First (0 min) and last (240 min) images from representative recordings are shown. In ...

Faciogenital Dysplasia Protein (FGD1) Regulates Export of Cargo Proteins from the Golgi Complex via Cdc42 Activation

“…In control cells, the formation of PGCs often occurs from long (2-10 m) tubular protrusions that bud from the Golgi complex (Figure 4, A and C, arrows; corresponding Movie 1), which is consistent with previous observations (Kreitzer et al, 2000;Polishchuk et al, 2003). In contrast, FGD1-deficient cells showed only short protrusions coming out from the Golgi complex, and these frequently retracted back into the Golgi without detachment of free transport carriers (Figure 4, B and D; arrows; corresponding Movie 2).…”

“…Moreover, even when PGCs did pinch off from the Golgi under RNAi, only a few of these moved in a centrifugal direction toward the cell surface; the others remained to hover around the Golgi area. A similar behavior of PGCs was seen in cells injected with an anti-kinesin antibody (Kreitzer et al, 2000). Therefore, FGD1 appears to be required for the interactions between these tubular TGN membranes and the cytoskeleton elements that are involved in their pulling out of, and later fission from, the Golgi complex.…”

The epithelial polarity program: machineries involved and their hijacking by cancer

“…Apical endocytosis requires polymerized actin, whereas basolateral endocytosis does not (Gottlieb et al, 1993). The exit of apical proteins from TGN/RE requires the actinand MT-associated protein dynamin 2 (Kreitzer et al, 2000;Bonazzi et al, 2005), whereas basolateral proteins require different proteins, such as BARS (Kreitzer et al, 2000;Bonazzi et al, 2005) and protein kinase D (Yeaman et al, 2004a).…”

Protein kinase D regulates basolateral membrane protein exit from trans-Golgi network

“…10) might be involved in sorting proteins in the TGN and/or endosomes [11][12][13] . Transport from the TGN to the apical plasma membrane is dependent on dynamin and a kinesin-like motor protein 14 . Cdc42 seems to regulate trafficking to both apical and basolateral membranes [15][16][17] , and assays in vitro for factors involved in vesicle production have revealed a requirement for an Arf-like GTPase, and activities of PKC-like and phospholipase D-like enzymes 18,19 .We examined roles of PKD isoforms in protein trafficking in Madin-Darby canine kidney (MDCK) cells.…”