The membrane transport factor TAP/p115 cycles between the Golgi and earlier secretory compartments and contains distinct domains required for its localization and function.
The mammalian protein TAP/p115 and its yeast homologue Uso1p have an essential role in membrane traffic (Nakajima et al., 1991; Waters et al., 1992; Sztul et al., 1993; Rabouille et al.; 1995). To inquire into the site and mechanism of TAP/p115 action, we aimed to localize it and to identify domains required for its function. We show that in interphase cells, TAP/p115 localizes predominantly to the Golgi and to peripheral structures that represent vesicular tubular clusters (VTCs) involved in ER to Golgi transport. Using BFA/ nocodazole treatments we confirm that TAP/p115 is present on ER to Golgi transport intermediates. TAP/ p115 redistributes to peripheral structures containing ERGIC-53 during a 15 degreesC treatment, suggesting that it is a cycling protein. Within the Golgi, TAP/p115 is associated with pleiomorphic structures on the cis side of the cis-Golgi cisterna and the cis-most cisterna, but is not detected in more distal compartments of the Golgi. TAP/p115 binds the cis-Golgi protein GM130, and the COOH-terminal acidic domain of TAP/p115 is required for this interaction. TAP/p115 interaction with GM130 occurs only in the Golgi and is not required for TAP/p115 association with peripheral VTCs. To examine whether interaction with GM130 is required to recruit TAP/p115 to the Golgi, TAP/p115 mutants lacking the acidic domain were expressed and localized in transfected cells. Mutants lacking the GM130-binding domain showed normal Golgi localization, indicating that TAP/p115 is recruited to the Golgi independently of its ability to bind GM130. Such mutants were also able to associate with peripheral VTCs. Interestingly, TAP/p115 mutants containing the GM130-binding domain but lacking portions of the NH2-terminal region were restricted from the Golgi and localized to the ER. The COOH-terminal domain required for GM130 binding and the NH2-terminal region required for Golgi localization appear functionally relevant since expression of TAP/p115 mutants lacking either of these domains leads to loss of normal Golgi morphology.
Nelson
D S
DS
Department of Cell Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Alvarez
C
C
Gao
Y S
YS
García-Mata
R
R
Fialkowski
E
E
Sztul
E
E
D000143:Acids / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001324:Autoantigens; D001692:Biological Transport / Q000502:physiology; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation / Q000502:physiology; D003594:Cytoplasmic Granules / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism* / Q000648:ultrastructure; D000077614:Golgi Matrix Proteins; D007668:Kidney / Q000166:cytology; D008099:Liver / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection; D033921:Vesicular Transport Proteins
D000143:Acids; D001324:Autoantigens; D002352:Carrier Proteins; D000077614:Golgi Matrix Proteins; C097741:Golgin subfamily A member 2; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C091580:vesicular transport factor p115