The Synaptojanin-like Protein Inp53/Sjl3 Functions with Clathrin in a Yeast TGN-to-Endosome Pathway Distinct from the GGA Protein-dependent Pathway

Abstract

Yeast TGN resident proteins that frequently cycle between the TGN and endosomes are much more slowly transported to the prevacuolar/late endosomal compartment (PVC) than other proteins. However, TGN protein transport to the PVC is accelerated in mutants lacking function of Inp53p. Inp53p contains a SacI polyphosphoinositide phosphatase domain, a 5-phosphatase domain, and a proline-rich domain. Here we show that all three domains are required to mediate "slow delivery" of TGN proteins into the PVC. Although deletion of the proline-rich domain did not affect general membrane association, it caused localization to become less specific. The proline-rich domain was shown to bind to two proteins, including clathrin heavy chain, Chc1p. Unlike chc1 mutants, inp53 mutants do not mislocalize TGN proteins to the cell surface, consistent with the idea that Chc1p and Inp53p act at a common vesicular trafficking step but that Chc1p is used at other steps also. Like mutations in the AP-1 adaptor complex, mutations in INP53 exhibit synthetic growth and transport defects when combined with mutations in the GGA proteins. Taken together with other recent studies, our results suggest that Inp53p and AP-1/clathrin act together in a TGN-to-early endosome pathway distinct from the direct TGN-to-PVC pathway mediated by GGA/clathrin.

A Role For Lte1p (a Low Temperature Essential Protein Involved in Mitosis) in Proprotein Processing in the Yeast Secretory Pathway

“…6). These results are compatible with a growing list of mutants in trafficking genes that affect Golgi-early endosomal trafficking and secretory proprotein processing yet do not produce a vps phenotype, such as tlg2 (and to a large extent, tlg1 (25), chc1 (26), as well as inp53 (27,28)). As one intracellular site of Vps21p function involves Golgi-early endosome trafficking (29), Lte1p may facilitate Vps21p activity at this site; thus lte1 should be considered a candidate for addition to the foregoing list of mutants.…”

The ZebrafishnrcMutant Reveals a Role for the Polyphosphoinositide Phosphatase Synaptojanin 1 in Cone Photoreceptor Ribbon Anchoring

“…The catalytic sites of the zebrafish SacI and 5Ј-phosphatase domains are highly conserved. Amphiphysin (Cestra et al, 1999), endophilin (Cestra et al, 1999;Ringstad et al, 2001), and putative clathrin binding domains (Ha et al, 2003) are also highly conserved (domains highlighted in Fig. 1).…”

Fab1p and AP-1 are required for trafficking of endogenously ubiquitylated cargoes to the vacuole lumen in S. cerevisiae

“…7). Although the exact role of AP-1 in trafficking to or from the TGN has yet to be elucidated, one current model describes two parallel clathrin-dependent pathways: the Golgiassociated gamma-ear-containing ADP-ribosylation-factorbinding protein (GGA)-dependent pathway and the AP-1-dependent pathway (Black and Pelham, 2000;Ha et al, 2003;Hinners and Tooze, 2003), the latter of which we propose also requires Fab1p and PtdIns(3,5)P 2 . GGA proteins bind and sort ubiquitylated cargoes, so it is possible that any cargoes ubiquitylated at the TGN will transit on the GGA-dependent route to the MVB (Pelham, 2004;Scott et al, 2004).…”

“…In S. cerevisiae there are three AP complexes: AP-1, AP-2 and AP-3 (Boehm and Bonifacino, 2001;Boehm and Bonifacino, 2002), of which only AP-1 has been shown to interact with clathrin: AP-1 complex gene disruptions exacerbate the defects seen in clathrin-heavy-chain mutants; and both Apl2p and Apl4p (the ␤-and ␥-adaptin subunits of the AP-1 complex, respectively) have been shown to bind clathrin (Phan et al, 1994;Rad et al, 1995;Stepp et al, 1995;Yeung and Payne, 2001;Yeung et al, 1999), whereas S. cerevisiae AP-2 and AP-3 seem to function in a clathrin-independent manner (Cowles et al, 1997;Yeung et al, 1999). Most recent data would support a role for AP-1 in clathrin-coated vesicle (CCV)-dependent bi-directional transport between the TGN and the early endosome (EE) (Hinners and Tooze, 2003), and in S. cerevisiae this function of AP-1 ensures the proper localization of several proteins including Kex2p, Chs3p and Tgl1p (Ha et al, 2003;Valdivia et al, 2002;Yeung and Payne, 2001;Yeung et al, 1999). However, many of the molecular details of AP-1-dependent trafficking have yet to be determined.…”